Combined organ and hematopoietic cells for transplantation tolerance of grafts

ABSTRACT

Methods and compositions are provided for combined transplantation of a solid organ and hematopoietic cells to a recipient, where tolerance to the graft is established through development of a persistent mixed chimerism. An individual with persistent mixed chimerism, usually for a period of at least six months, is able to withdraw from the use of immunosuppressive drugs after a period of time sufficient to establish tolerance.

CROSS-REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 16/100,828 filed Aug. 10, 2018, which is a Continuation ofapplication Ser. No. 15/914,746 filed Mar. 7, 2018, now U.S. Pat. No.10,076,542, issued Sep. 18, 2018, which is a Continuation of applicationSer. No. 15/387,292 filed Dec. 21, 2016, now U.S. Pat. No. 9,974,807,issued May 22, 2018, which is a Continuation of application Ser. No.14/438,159 filed Apr. 23, 2015, now U.S. Pat. No. 9,561,253 issued Feb.7, 2017, which is a 371 application and claims the benefit of PCTApplication No. PCT/US2014/015394, filed Feb. 7, 2014, which claimsbenefit of U.S. Provisional Application No. 61/769,596, filed Feb. 26,2013, which applications are incorporated herein by reference.

BACKGROUND

Advances in surgical techniques and improved drugs that preventinfection and rejection have allowed transplantation of solid organs tobecome an effective treatment for many diseases. Transplanted organsinclude heart, intestine, liver, lung, pancreas and kidney. Kidneytransplantation, or renal transplantation, is the organ transplant of akidney into a patient with end-stage renal disease. Kidneytransplantation can be classified as deceased or living-donortransplantation, and may further be classified according to the degreeof relationship between donor and recipient, as related or non-related,and according to the number of human leukocyte antigen (HLA)-matches orHLA-mismatches.

The indication for kidney transplantation is end-stage renal disease(ESRD), regardless of the primary cause, defined as a glomerularfiltration rate below a pre-determined level. Common diseases leading toESRD include malignant hypertension, infections, diabetes mellitus, andfocal segmental glomerulosclerosis; genetic causes include polycystickidney disease, a number of inborn errors of metabolism, and autoimmuneconditions such as lupus. Diabetes is a common cause of kidneytransplantation, accounting for approximately 25% of those in the US.The majority of renal transplant recipients are on dialysis at the timeof transplantation.

The major barrier to organ transplantation between geneticallynon-identical patients lies in the recipient's immune system, which canrespond to the transplanted kidney as “non-self” and reject it. Thus,having medications to suppress the immune system is essential, however,suppressing an individual's immune system places that individual atgreater risk of infection and cancer, in addition to the side effects ofthe medications. Recipients usually receive a mixture of threemaintenance immunosuppressive drugs, including a calcineurin inhibitorsuch as cyclosporine A, tacrolimus or sirolimus; prednisone; and aninhibitor of nucleic acid synthesis such as mycophenolate mofetil. Thelatter drugs have side effects that include hypertension,nephrotoxicity, infection, and heart disease that contribute to longterm patient disability and graft loss. In spite of modernimmunosuppressive drugs, in some centers acute rejection can occur in10-25% of people after transplant.

Generally transplant recipients will take immunosuppressiveanti-rejection drugs for as long as the transplanted kidney functions.Even for a mixture of widely used immunosuppressives the cost can behigh.

Preclinical studies have shown that conditioning with total lymphoidirradiation (TLI) and anti-thymocyte globulin (ATG) is advantageous forinducing tolerance after combined organ and bone marrow transplantationbecause the conditioning regimen prevents graft versus host disease(GVHD) as compared to total body irradiation (TBI). For a review, seeStrober et al. (2011) Seminars in Immunology 23:273-281.

It is therefore of great clinical interest to develop therapeuticregimens that achieve tolerance and complete withdrawal ofimmunosuppressive drugs in adult transplant patients and withoutinduction of GVHD.

SUMMARY

Methods and compositions are provided herein for the combinedtransplantation of a solid organ and hematopoietic cells to a recipient,where tolerance to the graft is established through development of astable mixed chimera. Preferably, the solid organ is a kidney. Anindividual with stable mixed chimerism, usually for a period of at leastsix months, is able to withdraw from the use of immunosuppressive drugsafter a period of time sufficient to establish tolerance.

Disclosed herein are methods for organ transplantation. The methodsprovided herein describe, in some cases, following transplantation of anHLA-matched or HLA-mismatched solid organ, administration ofdonor-derived hematopoietic stem cells to a recipient. In some cases,the donor-derived hematopoietic stem cells may be prepared to be atleast 70% pure prior to formulation as an engineered hematopoietic cellcomposition.

In some cases, a method for transplantation of an HLA-mismatched solidorgan from a donor comprising implanting the HLA-mismatched solid humanorgan from the donor in a recipient human body, treating the recipientwith non-myeloablative conditioning, infusing the recipient with anengineered hematopoietic cell composition comprising at least 1×10⁶CD34⁺ cells/kg and at least 1.0×10⁷ CD3⁺ cells/kg, and maintaining therecipient on an immunosuppressive regimen for a period of timesufficient to develop mixed chimerism for at least six months isdisclosed.

In some cases, the methods may include infusing at least 10×10⁶ CD34⁺cells/kg recipient weight and at least 1.0×10⁶ CD3⁺ cells/kg into therecipient. In some cases, at least 10×10⁶ CD34⁺ cells/kg recipientweight and at least 1.0×10⁷ CD3⁺ cells/kg are infused into therecipient. In some cases, at least 10×10⁶ CD34⁺ cells/kg recipientweight and between 1.0-5.0×10⁶ CD3⁺ cells/kg are infused into therecipient. In some cases, less than 15×10⁶ CD34⁺ cells/kg recipientweight and at least 50×10⁶ CD3⁺ cells/kg are infused into the recipient.

In some cases, a method is provided for transplantation of a solidorgan, the method comprising implanting the solid human organ from adonor in a recipient human body, and infusing the recipient with anallogeneic hematopoietic cell composition comprising donor-derived CD34⁺cells and CD3⁺ cells for a period of time sufficient to allow withdrawalfrom immunosuppressive drugs in said recipient for a period of at leastsix months is disclosed. For example, the cellular concentration of iron(e.g. bound to or internalized) in the donor-derived CD34⁺ cells and/orthe cellular concentration of iron in the donor-derived CD3⁺ cells maybe between 5 and 100 pg.

In some cases, a method is provided for transplantation of a solidorgan, the method comprising, implanting the solid human organ from adonor in a recipient human body; and infusing the recipient with anallogeneic hematopoietic cell composition comprising donor-derived CD34⁺cells of at least 70% purity and CD3⁺ cells for a period of timesufficient to allow withdrawal from immunosuppressive drugs in saidrecipient for a period of at least six months.

In some cases, the methods may include isolation of CD34+ cells from atleast one apheresis product, two apheresis products, three apheresisproducts, four apheresis products or five apheresis products. In somecases, the apheresis product is isolated from a solid organ donor. Insome cases, the CD3+ cells are isolated from a CD34-depleted fraction ofthe apheresis product. In some cases, the CD34-depleted fraction of theapheresis product is the CD34-depleted flow-through fraction from anaffinity column.

In some cases, the methods may include transplantation of a solid organfrom a donor to a recipient, wherein the solid organ is selected from agroup consisting of a heart, intestine, liver, lung, pancreas andkidney. The solid organ may be a portion of a whole organ, may beobtained from a living donor or a deceased donor and/or may be relatedor non-related to the recipient.

In some cases, the methods may include determining whether a donor and arecipient are HLA-matched or HLA-mismatched by typing the six HLAalleles of HLA-A, HLA-B and HLA-DR in each of the donor and therecipient. HLA-matched cells are those in which all six of the HLAalleles are the same between the donor and the recipient. HLA-mismatchedcells are those in which at least one HLA allele of the six is differentbetween the donor and the recipient.

In some cases, the methods may include that the recipient undergoesnon-myeloablative conditioning, which conditioning comprises lymphoidtissue irradiation in combination with T cell depleting antibodies ordrugs prior to infusing the cell composition. In some cases, the methodsmay include that the recipient undergoes an immunosuppressive regimenthat can include but is not limited to a calcineurin inhibitor, and apurine metabolism inhibitor for a period of at least six months.

In some cases, the methods may also include monitoring the recipient forstable mixed chimerism. Mixed chimerism is defined as an individualhaving at least 1% and less than 95% circulating donor hematopoieticand/or immune cells. In some cases, stable mixed chimerism is defined ashaving at least 1% and less than 10% circulating donor hematopoieticand/or immune cells, less than 15%, less than 20%, less than 25%, lessthan 30%, less than 35%, less than 40%, less than 45%, less than 50%,less than 55%, less than 60%, less than 65%, less than 70%, less than75%, less than 80%, less than 85% or less than 90% circulating donorhematopoietic and/or immune cells, for a period of time, for example forat least 1 month, at least 2 months, at least 3 months, at least 4months, at least 5 months at least 6 months.

In some cases, the methods may include withdrawal of immunosuppressionfrom individuals found to have mixed chimerism for at least threemonths, for at least six months or for at least twelve months.

Disclosed herein are compositions for use with organ transplantation.The compositions provided herein find use, in some cases, foradministration of a composition of donor-derived hematopoietic stemcells to a recipient following transplantation of an HLA-matched orHLA-mismatched solid organ. In some cases, such compositions comprisedonor-derived hematopoietic stem cells, which may be at least 70% pure.In some cases, the allogeneic hematopoietic cell composition comprisesdonor-derived CD34⁺ cells and donor-derived CD3⁺ cells, the cellularconcentration of iron in each of the donor-derived cells is between 5and 100 pg, wherein the number of cells is an amount sufficient to allowwithdrawal from immunosuppressive drugs when transplanted in a recipientat least one year following solid organ transplantation.

In some cases, the compositions may include infusion of at least 10×10⁶CD34⁺ cells/kg recipient weight and at least 1.0×10⁶ CD3⁺ cells/kg intothe recipient. In some cases, at least 10×10⁶ CD34⁺ cells/kg recipientweight and at least 1.0×10⁷ CD3⁺ cells/kg into the recipient. In somecases, at least 10×10⁶ CD34⁺ cells/kg recipient weight and between1.0-5.0×10⁶ CD3⁺ cells/kg into the recipient. In some cases, less than15×10⁶ CD34⁺ cells/kg recipient weight and at least 50×10⁶ CD3⁺ cells/kginto the recipient.

In some cases, the solid organ is HLA-matched or HLA-mismatched. In somecases, the solid organ is selected from a group consisting of a heart,intestine, liver, lung, pancreas and kidney.

In some cases, the composition may be administered along withtransplantation of a solid organ from a donor to a recipient, whereinthe solid organ is selected from a group consisting of a heart,intestine, liver, lung, pancreas and kidney. The solid organ may be aportion of a whole organ, may be obtained from a living donor or adeceased donor and/or may be related or non-related to the recipient.

In some cases, the compositions may be administered after determiningwhether a donor and a recipient are HLA-matched or HLA-mismatched bytyping HLA alleles HLA-A, HLA-B and HLA-DR in the donor and therecipient. HLA-matched may refer to a match wherein each of the HLAalleles at HLA-A, HLA-B and HLA-DR are the same between the donor andthe recipient. HLA-mismatched may refer to a match wherein at least oneHLA allele at HLA-A, HLA-B and HLA-DR is different between the donor andthe recipient.

In some cases, a composition of isolated hematopoietic cells formulatedfor administration to a recipient may comprise at least 1×10⁶/kgintended recipient of CD34⁺ cells isolated from a donor-derivedapheresis product, wherein a cellular concentration of iron in the CD34⁺cells is between 5 and 100 pg of iron per cell, and at least 1×10⁷/kgintended recipient of CD3⁺ T cells isolated from a donor-derivedapheresis product, wherein a cellular concentration of iron in the CD3⁺cells is between 5 and 100 pg of iron per cell, and a pharmaceuticalcarrier. Weight of an intended recipient may be determined prior toformulation of the engineered product. Alternatively a product can beformulated with the appropriate number of or ratio of CD34⁺ cells andCD3⁺ cells, and the appropriate volume of the product administered tothe recipient.

In some cases, the composition may be generated from an apheresisproduct that is derived from a donor treated with at least 10micrograms/kg of G-CSF. In some cases, the G-CSF is administered to thedonor in two doses. In some cases, the apheresis product is isolatedfrom the donor less than five hours after the second dose of G-CSF.

In some cases, the composition may include CD34+ cells and CD3+ cellsisolated from the apheresis product using at least an affinity agent anda column, the CD34+ cells located in an eluate from the column and theCD3+ cells located in a CD34+ cell depleted flow-through fraction fromthe column.

In some cases, the cellular concentration of iron in the CD34⁺ cells inthe composition is between 5 and 100 pg per cell, for example 5 and 10pg of iron per cell, 7.5 and 15 pg of iron per cell, 10 and 20 pg ofiron per cell, 15 and 30 pg of iron per cell, 20 and 40 pg of iron percell, 30 and 50 pg of iron per cell, 40 and 60 pg of iron per cell, 50and 70 pg of iron per cell, 60 and 80 pg of iron per cell, 70 and 90 pgof iron per cell or 80 and 100 pg of iron per cell.

In some cases, the cellular concentration of iron in the CD3⁺ cells inthe composition is between 5 and 10 pg of iron per cell, 7.5 and 15 pgof iron per cell, 10 and 20 pg of iron per cell, 15 and 30 pg of ironper cell, 20 and 40 pg of iron per cell, 30 and 50 pg of iron per cell,40 and 60 pg of iron per cell, 50 and 70 pg of iron per cell, 60 and 80pg of iron per cell, 70 and 90 pg of iron per cell or 80 and 100 pg ofiron per cell.

In some cases, the CD34+ cells of the hematopoietic cell composition areat least 70% pure prior to formulation of the hematopoietic cellcomposition. In some cases, the composition is administered to arecipient that is HLA-matched to the CD34+ cells and CD3+ cells, orHLA-mismatched to the CD34+ cells and CD3+ cells. In some cases, thecells of the composition are determined to be HLA-matched orHLA-mismatched by typing HLA alleles HLA-A, HLA-B, and HLA-DR in thedonor and the recipient.

In some cases, a cellular composition is generated from hematopoieticcells which are HLA-matched, where HLA alleles HLA-A, HLA-B, HLA-DR arethe same between the donor and the recipient. In some cases, thecomposition is generated from hematopoietic cells which areHLA-mismatched, where at least one HLA allele of HLA-A, HLA-B, HLA-DR isdifferent between the donor and the recipient. In some cases, thecomposition is generated from an apheresis product taken from a donorthat is related or non-related to the recipient.

In some cases, the compositions may include CD34+ cells that areisolated from at least one apheresis product, two apheresis products,three apheresis products, four apheresis products or five apheresisproducts. In some cases, the apheresis product is isolated from a solidorgan donor. In some cases, the CD3+ cells are isolated from aCD34-depleted fraction of the apheresis product. In some cases, theCD34-depleted fraction of the apheresis product is the CD34-depletedflow-through fraction from an affinity column.

Disclosed herein are kits for organ transplantation. The kits providedherein describe, in some cases, preparation of a composition ofhematopoietic stem cells which may be administered to a recipientfollowing transplantation of an HLA-matched or HLA-mismatched solidorgan. In some cases, the kit of component parts capable for use inpreparing an engineered hematopoietic cell composition comprises atleast one affinity reagent for CD34, at least one device for separatingCD34+ cells from a plurality of cells, at least one agent fordetermining a number of CD3+ cells; and instructions for isolating CD34+cells from a plurality of cells and determining the number of CD3+ cellsusing at least the component parts of the kit to generate the engineeredhematopoietic cell composition.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 illustrates a protocol for kidney and hematopoietic celltransplantation for a haplotype matched donor and recipient.

FIG. 2 is a table of results from haplotype matched organ transplants.

FIG. 3 provides a graph assessment of chimerism following haplotypematched combined organ transplant in patient #2 from FIG. 2.

FIG. 4A-4D provide a graph assessment of chimerism for 4 differentrecipients. Each recipient is shown in one of panels FIG. 4A, FIG. 4B,FIG. 4C and FIG. 4D. The number of CD34+ and CD3+ cells administered tothe recipient is shown above the graph.

DETAILED DESCRIPTION

While preferred aspects of the present disclosure have been shown anddescribed herein, it is to be understood that the disclosure is notlimited to the particular aspects of the disclosure described below, asvariations of the particular aspects may be made and still fall withinthe scope of the appended claims. It is also to be understood that theterminology employed is for the purpose of describing particular aspectsof the disclosure, and is not intended to be limiting. Instead, thescope of the present disclosure is established by the appended claims.In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise.

Definitions

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure provided herein. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe disclosure provided herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the disclosure, the preferredmethods, devices and materials are now described.

“Major histocompatibility complex antigens” (“MHC”, also called “humanleukocyte antigens”, HLA) are protein molecules expressed on the surfaceof cells that confer a unique antigenic identity to these cells. MHC/HLAantigens are target molecules that are recognized by T-cells and naturalkiller (NK) cells as being derived from the same source of hematopoieticstem cells as the immune effector cells (“self”) or as being derivedfrom another source of hematopoietic reconstituting cells (“non-self”).Two main classes of HLA antigens are recognized: HLA class I and HLAclass II. HLA class I antigens (A, B, and C in humans) render each cellrecognizable as “self,” whereas HLA class II antigens (DR, DP, and DQ inhumans) are involved in reactions between lymphocytes and antigenpresenting cells. Both have been implicated in the rejection oftransplanted organs.

An important aspect of the HLA gene system is its polymorphism. Eachgene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR) existsin different alleles. HLA alleles are designated by numbers andsubscripts. For example, two unrelated individuals may carry class IHLA-B, genes B5, and Bw41, respectively. Allelic gene products differ inone or more amino acids in the α and/or β domain(s). Large panels ofspecific antibodies or nucleic acid reagents are used to type HLAhaplotypes of individuals, using leukocytes that express class I andclass II molecules. The genes most important for HLA typing are the sixMHC Class I and Class II proteins, two alleles for each of HLA-A; HLA-Band HLA-DR.

The HLA genes are clustered in a “super-locus” present on chromosomeposition 6p21, which encodes the six classical transplantation HLA genesand at least 132 protein coding genes that have important roles in theregulation of the immune system as well as some other fundamentalmolecular and cellular processes. The complete locus measures roughly3.6 Mb, with at least 224 gene loci. One effect of this clustering isthat “haplotypes”, i.e. the set of alleles present on a singlechromosome, which is inherited from one parent, tend to be inherited asa group. The set of alleles inherited from each parent forms ahaplotype, in which some alleles tend to be associated together.Identifying a patient's haplotypes can help predict the probability offinding matching donors and assist in developing a search strategy,because some alleles and haplotypes are more common than others and theyare distributed at different frequencies in different racial and ethnicgroups.

As used herein, the term “HLA matched” refers to a donor recipient pairin which none of the HLA antigens are mismatched between the donor andrecipient. HLA matched (i.e., where all of the 6 alleles are matched)donor/recipient pairs have a decreased risk of graft v. host disease(GVHD) relative to mismatched pairs (i.e. where at least one of the 6alleles is mismatched).

As used herein, the term “HLA mismatched” refers to a donor recipientpair in which at least one HLA antigen, in particular with respect toHLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient.In some cases, one haplotype is matched and the other is mismatched.This situation is frequently found with organs from living or deceaseddonors. HLA mismatched donor/recipient pairs have an increased risk ofGVHD relative to perfectly matched pairs (i.e. where all 6 alleles arematched).

HLA alleles are typically noted with a variety of levels of detail. Mostdesignations begin with HLA- and the locus name, then * and some (even)number of digits specifying the allele. The first two digits specify agroup of alleles. Older typing methodologies often could not completelydistinguish alleles and so stopped at this level. The third throughfourth digits specify a synonymous allele. Digits five through sixdenote any synonymous mutations within the coding frame of the gene. Theseventh and eighth digits distinguish mutations outside the codingregion. Letters such as L, N, Q, or S may follow an allele's designationto specify an expression level or other non-genomic data known about it.Thus, a completely described allele may be up to 9 digits long, notincluding the HLA-prefix and locus notation.

As used herein, a “recipient” is an individual to whom an organ, tissueor cells from another individual (donor), commonly of the same species,has been transferred. For the purposes of the present disclosure, arecipient and a donor are either HLA-matched or HLA-mismatched.

As used herein, the term “solid organ transplantation” is used inaccordance with the conventional meaning of the term, where an organfrom a donor, which donor may be living or deceased, in placed into thebody of a recipient in the appropriate position and cardiovascularconnections to be physiologically integrated into the recipient.Transplantation of a kidney is of particular interest for the methods ofthe disclosure, although the methods do not exclude transplantation ofother organs, e.g. pancreas and including pancreatic islet cells; heart;lungs, intestine, liver, and the like as known in the art. Thetransplanted organ may be referenced as a “graft”, and the physiologicalintegration of the organ may be referred to as engraftment.

Hematopoietic stem cell transplantation (HCT) is the transplantation ofmultipotent hematopoietic stem cells, usually derived from bone marrow,peripheral blood, or umbilical cord blood. For the methods of thedisclosure, the hematopoietic cells may be engineered into one of twoproducts. The hematopoietic cells are engineered into a product forinfusion having a specific pre-determined number of purified (e.g., ≥70%purity) CD34+ progenitor cells and CD3+ T cells. The hematopoietic cellscan be obtained from the solid organ donor, and thus are HLA-matched tothe solid organ, and HLA-mismatched to the organ recipient. Thehematopoietic cells may be obtained from the solid organ donor, and thusare HLA-matched to the solid organ, and HLA-matched to the organrecipient.

Where the donor is deceased, hematopoietic cells may be obtained frombone marrow (e.g. vertebrae, pelvic bone, etc). Where the donor is aliving donor, hematopoietic cells may be mobilized (e.g. with G-CSF),and collected by apheresis or similar methods. Alternatively, cells maybe obtained from bone marrow (e.g. pelvic bone, etc).

Hematopoietic cells can be frozen (e.g., cryopreserved) for prolongedperiods without damaging a significant number of cells. To cryopreserveHSC, a preservative, DMSO, must be added, and the cells must be cooledvery slowly in a controlled-rate freezer to prevent osmotic cellularinjury during ice crystal formation. HSC may be stored for years in acryofreezer, which typically uses liquid nitrogen.

The recipient's immune system is conditioned with a non-myeloablativeprocedure prior to infusion of the hematopoietic cells.Non-myeloablative transplants use doses of antibody and radiation thatare too low to eradicate all the bone marrow cells of a recipient, thusenabling the desired goal of stable mixed chimerism where both recipientand donor HSC coexist in the bone marrow space. The conditioning regimenincludes treatment with anti-thymocyte globulin (ATG); total lymphoidirradiation, and corticosteroids (e.g. prednisone) usually for a periodof from about 10 to 12 days (e.g. for about 11 days).

“Immunosuppression”, as used herein, refers to the treatment of a graftrecipient with agents, primarily to diminish the immune responses of thehost immune system against the graft, although the agents may alsodiminish GVHD of the donor hematopoietic cells. Exemplaryimmunosuppression regimens are described in more detail herein, but willgenerally be conventional for a period of about 6 to 12 months. Therecipient is tested for mixed chimerism of the hematopoietic system, andif found to have maintained mixed chimerism after at least 6 months,will be tapered off immunosuppression.

Immunosuppressive treatment of the transplantation patient begins withthe induction phase, perioperatively and immediately aftertransplantation. Maintenance therapy then continues until withdrawal forindividuals showing stable mixed chimerism. Induction and maintenancestrategies use different medicines at specific doses or at dosesadjusted to achieve target therapeutic levels to give thetransplantation patient the best hope for long-term graft survival.

Primary immunosuppressive agents include calcineurin inhibitors, whichcombine with binding proteins to inhibit calcineurin activity, and whichinclude, for example, tacrolimus, cyclosporine A, etc. Levels of bothcyclosporine and tacrolimus must be carefully monitored. Initially,levels can be kept in the range of 10-20 ng/mL, but, after 3 months,levels may be kept lower (5-10 ng/mL) to reduce the risk ofnephrotoxicity.

Adjuvant agents are usually combined with a calcineurin inhibitor andinclude steroids, azathioprine, mycophenolate mofetil, and sirolimus.Protocols of interest include a calcineurin inhibitor with mycophenolatemofetil. The use of adjuvant agents allows clinicians to achieveadequate immunosuppression while decreasing the dose and toxicity ofindividual agents. Mycophenolate mofetil in kidney transplant recipientshas assumed an important role in immunosuppression after severalclinical trials have shown a markedly decreased prevalence of acutecellular rejection compared with azathioprine and a reduction in 1-yeartreatment failures.

Antibody-based therapy uses monoclonal (e.g., muromonab-CD3) orpolyclonal antibodies or anti-CD25 antibodies (e.g., basiliximab,daclizumab) and is administered in the early posttransplant period (upto 8 wk). Antibody-based therapy allows for avoidance or dose reductionof calcineurin inhibitors, possibly reducing the risk of nephrotoxicity.The adverse effect profile of the polyclonal and monoclonal antibodieslimits their use in some patients.

Graft-versus-host disease (GVHD) is an inflammatory disease that ispeculiar to transplantation of hematopoietic cells. It is an attack ofthe donor bone marrow's immune cells against the recipient's tissues.GVHD is a risk for both HLA-matched and -mismatched transplantations.GVHD can occur even if the donor and recipient are HLA-matched becausethe immune system can still recognize other differences between theirtissues. GVHD is usually mediated by T cells, which react to foreignpeptides presented on the MHC of the host. The risk of GVHD is markedlyreduced in patients with mixed instead of complete chimerism andachieving mixed chimerism is desirable for this reason. In addition,immunodeficiency and infection are more frequently observed in completeversus mixed chimerism.

There are two types of GVHD, acute and chronic. Acute GVHD typicallyoccurs in the first 3 months after transplantation and may involve theskin, intestine, or the liver. High-dose corticosteroids such asprednisone are a standard treatment.

Chronic GVHD may also develop after haplotype matched transplant andtypically occurs after the first 3 months following transplant. It isthe major source of late treatment-related complications, although itless often results in death. In addition to inflammation, chronic GVHDmay lead to the development of fibrosis, or scar tissue, similar toscleroderma; it may cause functional disability and require prolongedimmunosuppressive therapy.

“Acute transplant rejection” is the rejection by the immune system of atransplanted organ. Acute rejection is characterized by infiltration ofthe transplanted tissue by immune cells of the recipient, which carryout their effector function and destroy the transplanted tissue. Theonset of acute rejection is rapid and generally occurs in humans withina few weeks after transplant surgery.

Generally, acute rejection is inhibited or suppressed withimmunosuppressive drugs. Steroids are the mainstay of therapy for acuterejection episodes. The typical dosage is 3-5 mg/kg/d for 3-5 days,which is then tapered to a maintenance dose. ATG and muromonab-CD3 alsofind use.

“Chronic transplant rejection” generally occurs in humans within severalmonths to years after engraftment, even in the presence of successfulimmunosuppression of acute rejection. Fibrosis is a common factor inchronic rejection of all types of organ transplants. Chronic rejectioncan typically be described by a range of specific disorders that arecharacteristic of the particular organ. For example, in lungtransplants, such disorders include fibroproliferative destruction ofthe airway (bronchiolitis obliterans); in heart transplants ortransplants of cardiac tissue, such as valve replacements, suchdisorders include fibrotic atherosclerosis; in kidney transplants, suchdisorders include, obstructive nephropathy, nephrosclerorsis,tubulointerstitial nephropathy; and in liver transplants, such disordersinclude disappearing bile duct syndrome.

Chronic rejection can also be characterized by ischemic insult,denervation of the transplanted tissue, hyperlipidemia and hypertensionassociated with immunosuppressive drugs. Unless inadequateimmunosuppression is the cause of rejection, changes inimmunosuppressive therapy are generally not effective in reversingchronic rejection. Control of blood pressure, treatment ofhyperlipidemia, and management of diabetes are the current mainstays oftreatment for graft preservation.

The term “transplant rejection” encompasses both acute and chronictransplant rejection. In transplant rejection, the transplanted tissueis rejected and destroyed by the recipient's immune system. Acuterejection may occur to some degree in all transplants, except in thecases of identical twins or during immunosuppression. Acute rejectionmay begin as soon as one week after transplant and greatest risk fordevelopment of acute rejection occurs in the first three monthsfollowing transplant. Chronic rejection is the long-term loss offunction of a transplanted organ.

Hematopoietic cell transplant loss is the absence of hematopoieticreconstitution of donor origin on day +45 after the allograft (primarygraft rejection) or as confirmed loss of donor cells after transientengraftment of donor-origin hematopoiesis. Kidney graft failure iscreatinine clearance declining to less than 10 ml/min or the return ofthe patient to dialysis, or the return of the patient to the transplantlist for re-transplantation.

Chimerism, as used herein, generally refers to chimerism of thehematopoietic system, unless otherwise noted. A determination of whetheran individual is a full chimera, mixed chimera, or non-chimeric made bemade by an analysis of a hematopoietic cell sample from the graftrecipient, e.g. peripheral blood, bone marrow, etc. as known in the art.Analysis may be done by any convenient method of typing. In someembodiments the degree of chimerism amongst all mononuclear cells, Tcells, B cells, CD56+NK cells, and CD15+ neutrophils is regularlymonitored, using PCR with probes for microsatellite analysis. Forexample, commercial kits that distinguish polymorphisms in shortterminal repeat lengths of donor and host origin are available.Automated readers provide the percentage of donor type cells based onstandard curves from artificial donor and host cell mixtures.

Individuals who exhibited more than a 95% donor cells in a given bloodcell lineage by such analysis at any time post-transplantation arereferred to as having full donor chimerism in this transplant patientgroup. Mixed chimerism is defined as greater than 1% donor but less than95% donor DNA in such analysis. Individuals who exhibit mixed chimerismmay be further classified according to the evolution of chimerism, whereimproving mixed chimerism is defined as a continuous increase in theproportion of donor cells over at least a 6-month period. Stable mixedchimerism is defined as fluctuations in the percentage of recipientcells over time, without complete loss of donor cells. Candidates forwithdrawal of immunosuppression have mixed chimerism until at least 6months post-transplantation.

“Diagnosis” as used herein generally includes determination of asubject's susceptibility to a disease or disorder, determination as towhether a subject is presently affected by a disease or disorder,prognosis of a subject affected by a disease or disorder (e.g.,identification of pre-metastatic or metastatic cancerous states, stagesof cancer, or responsiveness of cancer to therapy), and use oftherametrics (e.g., monitoring a subject's condition to provideinformation as to the effect or efficacy of therapy).

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.

“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

The term “graft management” refers to therapeutic methods that induceand/or promote repair engraftment of a solid organ, but not limited to,kidney transplantation.

The term “pharmaceutically acceptable” as used herein refers to acompound or combination of compounds that will not impair the physiologyof the recipient human or animal to the extent that the viability of therecipient is compromised. Preferably, the administered compound orcombination of compounds will elicit, at most, a temporary detrimentaleffect on the health of the recipient human or animal.

The term “carrier” as used herein refers to any pharmaceuticallyacceptable solvent of agents that will allow a therapeutic compositionto be administered directly to a wound of the skin. The carrier willallow a composition to be topically applied to an exposed surface of anorgan for transplantation and the site of the recipient where the organis to be placed. A “carrier” as used herein, therefore, refers to suchsolvent as, but not limited to, water, saline, oil-water emulsions, orany other solvent or combination of solvents and compounds known to oneof skill in the art that is pharmaceutically and physiologicallyacceptable to the recipient human or animal.

The term “assessing” and “evaluating” are used interchangeably to referto any form of measurement, and includes determining if an element ispresent or not. The terms “determining,” “measuring,” “assessing,” and“assaying” are used interchangeably and include both quantitative andqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

Methods

The methods of the disclosure are discussed in detail below. In somecases, the methods described herein may comprise the steps of: HLAtyping a donor and recipient to determine an HLA-matched orHLA-mismatched pair. “HLA-matched” indicates all of the 6 HLA antigens(e.g., HLA-A, B, DR) are matched between a donor and a recipient.“HLA-mismatched” indicates that at least 1, at least 2, at least 3 of 6HLA antigens (e.g., HLA-A, B, DR) are mismatched. Generally at least aportion of the 6 HLA antigens (e.g., HLA-A, B, DR) are matched, forexample at least 1, at least 2, at least 3, at least 4, at least 6matches.

In some cases, the methods may include at least the following steps;obtaining the solid organ and hematopoietic cells from the donor;isolating hematopoietic cells of the appropriate type and dose;transplanting the solid organ; performing a conditioning regimen on therecipient following transplantation of the solid organ and prior toinfusion of the engineered hematopoietic cells; maintaining therecipient on an immunosuppressive regimen for at least six months;monitoring the recipient for mixed chimerism of the hematopoieticsystem; and withdrawing immunosuppression if the recipient shows stablemixed chimerism. The methods described herein apply to both HLA-matchedand HLA-mismatched transplantation conditions.

Individuals selected for the methods described herein may meet thecriteria of (i) requiring a solid organ graft; and (ii) having either anHLA-matched or HLA-mismatched donor from which the solid organ andhematopoietic cells can be obtained. By performing a combined transplantof solid organ and an engineered hematopoietic cell infusion appropriatefor the individual, in combination with non-myeloablative conditioning,the patient may have a high probability of developing persistent mixedchimerism for at least 6 months. Mixed chimerism which persists for atleast 6 months may allow for withdrawal of immunosuppression over time.

Typing Human Leukocyte Antigens

Any method known in the art may be used to type donor-derived cells anda sample from the recipient. For example, three main procedures may beused to perform HLA typing. The first is conventional serologicalcytotoxicity method, where samples of lymphocytes (e.g., taken from fromblood or spleen) are added to Terasaki plates. In some cases, Blymphocytes may be used for class II typing. In other cases, class Ityping may be performed with the remaining leucocytes. Magnetic beadsmay be used to purify cells from blood or spleen.

In some cases, each of the wells of the Terasaki plates may contain aplurality of antibodies (e.g., from either maternal sera or manufacturedmonoclonal antibodies). In some cases, the HLA antigen expressed by acell binds to an antibody in the well. After the addition of complement,cells located in a well where the HLA antigen and antibody were boundmay be killed. In some cases, a pattern of cell death may be determinedfrom the wells. The pattern may allow for deduction of the combinationof HLA antigens that were present on the original tissue. In some cases,the deduction of the combination of HLA antigens may result in typing ofHLA antigens.

Another method that may be used for HLA typing is flow cytometry. Unlikethe conventional serological cytotoxicity method, flow cytometry may beused to identify one or more HLA alleles. In this method, leukocytes maybe combined with antibodies that bind to the HLA types of interest. Insome cases the antibodies may be monoclonal or polyclonal. In somecases, the antibodies may contain a detectable label. In some cases, theantibodies may be directly conjugated to a detectable label. In othercases, a different antibody with a detectable label binds to the HLAantibody and the complex is then detected. The types of detectablelabels that may be used for HLA typing by flow cytometry are readilyavailable and known to those of skill in the art. The sample may beanalyzed to determine which HLA antibodies have bound to the cells.

Yet another method that may be used for HLA typing is DNA typing. Insome cases, DNA typing involves extracting DNA from cells and amplifyingthe genes that encode for the HLA peptides using polymerase chainreaction techniques which generate sequence data. The polymerase chainreaction techniques may include any polymerase chain reaction techniquewhich generates sequence data that is known to one of skill in the art.

In some cases, the sequence of the genes may be matched with the knownnucleotide sequences of HLA alleles located in at least one of severalgenetic (e.g., gene bank) databases. In some cases, the gene bank database may be the IMGT/HLA (International Immunogenetics Project)database.

Solid Organ Transplant

Solid organs may be transplanted from a donor to a recipient such thatthe organ is placed into the appropriate position in the recipient body.In some cases, the cardiovascular connections between the solid organmay be physiologically integrated into the recipient body. In somecases, the organ may be from a living donor. In other cases, the organmay be from a deceased donor. In some cases, the solid organ may beHLA-matched between the donor and the recipient. In other cases, thesolid organ may be HLA-mismatched between the donor and the recipient.

Any solid organ that may be used for organ transplantation may be usedwith the methods described herein. In some cases, the organ may be akidney, lung, pancreas, pancreatic islet cells, heart, intestine, colon,liver, skin, muscle, gum, eye, tooth and the like as known to those ofskill in the art. In some cases, the organ may be a complete organ. Inother cases, the organ may be a portion of an organ. In other cases, theorgan may be cells from a tissue of an organ.

Using the methods described herein, the solid organ is harvested andtransplanted in accordance with conventional practice. In some cases,the solid organ may be transplanted at least one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen or at least twenty daysprior to the infusion of the engineered hematopoietic cells.

Obtaining Hematopoietic Stem Cells for Transplantation

Hematopoietic stem cell transplantation (HCT) includes thetransplantation of multipotent hematopoietic stem cells from a donor toa recipient. For the methods described herein, HCT may be combined withsolid organ transplant. In some cases, the hematopoietic stem cells maybe HLA-matched between the donor and the recipient. In other cases, thehematopoietic stem cells may be HLA-mismatched between the donor and therecipient.

In some cases, the hematopoietic stem cells are isolated and purifiedfrom the solid organ donor. The solid organ donor may be living ordeceased. In cases of a living donor, hematopoietic cells may beobtained from the solid organ donor using any of the various methodsknown to one of skill in the art, including apheresis of mobilizedperipheral blood from living donors; harvesting hematopoietic cells frombone marrow of deceased donors, and the like. In cases of a deceaseddonor, hematopoietic cells may be obtained from bone marrow. Forexample, the cells may be obtained from the bone marrow in vertebrae,pelvic bone, femur or any other bone which contains sufficient bonemarrow from which to extract hematopoietic cells.

In some cases, hematopoietic cells may be mobilized prior to isolationand purification. In some cases hematopoietic cells may be mobilized bytreating the donor with granulocyte colony stimulating factor (G-CSF).For example, the donor may be treated with one, two, three, four, five,six, seven, eight, nine, ten or more than ten doses of G-CSF prior toisolating and purifying hematopoietic cells.

In some cases, the doses of G-CSF may be delivered to the donor on asingle day (e.g., a 24 hour day) or over the course of multiple days.For example, multiple days may include two, three, four, five, six,seven, eight, nine, ten or more than ten days. In a preferred case, thedonor receives two doses per day.

In some cases, each dose of G-CSF delivered to the donor is 16micrograms/kg of donor body weight. In other cases, each dose of G-CSFdelivered to the donor is 8 micrograms/kg of donor body weight. Forexample, each dose of G-CSF may be more than 1 micrograms/kg of donorbody weight, 2 micrograms/kg of donor body weight, 3 micrograms/kg ofdonor body weight, 4 micrograms/kg of donor body weight, 5 micrograms/kgof donor body weight, 6 micrograms/kg of donor body weight, 7micrograms/kg of donor body weight, 8 micrograms/kg of donor bodyweight, 9 micrograms/kg of donor body weight, 10 micrograms/kg of donorbody weight, 11 micrograms/kg of donor body weight, 12 micrograms/kg ofdonor body weight, 13 micrograms/kg of donor body weight, 14micrograms/kg of donor body weight, 15 micrograms/kg of donor bodyweight, 16 micrograms/kg of donor body weight, 17 micrograms/kg of donorbody weight, 18 micrograms/kg of donor body weight, 19 micrograms/kg ofdonor body weight, 20 micrograms/kg of donor body weight, or more than20 micrograms/kg of donor body weight. In a preferred case, each dose ofG-CSF delivered to the donor is 8 micrograms/kg of donor body weight.

In some cases, apheresis may be performed after the donor receives asingle dose of G-CSF. For example, apheresis may be performed one hour,two hours, three hours, four hours, five hours, six hours, seven hours,eight hours, nine hours, ten hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, ormore than 48 hours after the donor receives the single dose of G-CSF.

In some cases, apheresis may be performed after the donor receives thefinal dose of multiple doses of G-CSF. For example, apheresis may beperformed one hour, two hours, three hours, four hours, five hours, sixhours, seven hours, eight hours, nine hours, ten hours, 11 hours, 12hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47hours, 48 hours, or more than 48 hours after the donor receives thefinal dose of multiple doses of G-CSF.

In some cases, apheresis may be performed to obtain an apheresis productfrom a donor. For example, at least one apheresis product, two apheresisproducts, three apheresis products, four apheresis products or fiveapheresis products may be obtained from a donor. In some cases, at leasttwo apheresis products, three apheresis products, four apheresisproducts, five apheresis products, six apheresis products, sevenapheresis products, eight apheresis products, nine apheresis products,ten apheresis products, 11 apheresis products, 12 apheresis products, 13apheresis products, 14 apheresis products or at least 15 apheresisproducts may be obtained from a donor.

In some cases, the hematopoietic cells may be obtained from a solidorgan donor HLA-matched to the recipient. In this case, thehematopoietic cells are HLA-matched to the solid organ and the solidorgan recipient. In some cases, the hematopoietic cells may be obtainedfrom a solid organ donor HLA-mismatched to the recipient. In this case,the hematopoietic cells are HLA-matched to the solid organ andHLA-mismatched to the solid organ recipient.

For the methods described herein, hematopoietic cells may be frozen(e.g., cryopreserved) after isolation or after isolation andpurification from the solid organ donor. In some cases, hematopoieticcells may be cryopreserved using a cryopreservation medium and a methodof cryopreservation known to those of skill in the art. In some cases,the hematopoietic cells may be cryopreserved using a cryopreservationmedium containing dimethylsulfoxide (DMSO), Normosol, Hetastarch andhuman serum albumin (HSA).

In some cases, the concentration of DMSO in the cryopreservation mediummay be less than 0.1% DMSO, 0.2% DMSO, 0.3% DMSO, 0.4% DMSO, 0.5% DMSO,0.6% DMSO, 0.7% DMSO, 0.8% DMSO, 0.9% DMSO, 1.0% DMSO, 1.1% DMSO, 1.2%DMSO, 1.3% DMSO, 1.4% DMSO, 1.5% DMSO, 1.6% DMSO, 1.7% DMSO, 1.8% DMSO,1.9% DMSO, 2.0% DMSO, 2.1% DMSO, 2.2% DMSO, 2.3% DMSO, 2.4% DMSO, 2.5%DMSO, 2.6% DMSO, 2.7% DMSO, 2.8% DMSO, 2.9% DMSO, 3.0% DMSO, 3.1% DMSO,3.2% DMSO, 3.3% DMSO, 3.4% DMSO, 3.5% DMSO, 3.6% DMSO, 3.7% DMSO, 3.8%DMSO, 3.9% DMSO, 4.0% DMSO, 4.1% DMSO, 4.2% DMSO, 4.3% DMSO, 4.4% DMSO,4.5% DMSO, 4.6% DMSO, 4.7% DMSO, 4.8% DMSO, 4.9% DMSO, 5.0% DMSO, 5.1%DMSO, 5.2% DMSO, 5.3% DMSO, 5.4% DMSO, 5.5% DMSO, 5.6% DMSO, 5.7% DMSO,5.8% DMSO, 5.9% DMSO, 6.0% DMSO, 6.1% DMSO, 6.2% DMSO, 6.3% DMSO, 6.4%DMSO, 6.5% DMSO, 6.6% DMSO, 6.7% DMSO, 6.8% DMSO, 6.9% DMSO, 7.0% DMSO,7.1% DMSO, 7.2% DMSO, 7.3% DMSO, 7.4% DMSO, 7.5% DMSO, 7.6% DMSO, 7.7%DMSO, 7.8% DMSO, 7.9% DMSO, 8.0% DMSO, 8.1% DMSO, 8.2% DMSO, 8.3% DMSO,8.4% DMSO, 8.5% DMSO, 8.6% DMSO, 8.7% DMSO, 8.8% DMSO, 8.9% DMSO, 9.0%DMSO, 9.1% DMSO, 9.2% DMSO, 9.3% DMSO, 9.4% DMSO, 9.5% DMSO, 9.6% DMSO,9.7% DMSO, 9.8% DMSO, 9.9% DMSO, 10% DMSO, 10.5% DMSO, 11% DMSO, 11.5%DMSO, 12% DMSO, 12.5% DMSO, 13% DMSO, 13.5% DMSO, 14% DMSO, 14.5% DMSO,15% DMSO, 15.5% DMSO, 16% DMSO, 16.5% DMSO, 17% DMSO, 17.5% DMSO, 18%DMSO, 18.5% DMSO, 19% DMSO, 20% DMSO, 20.5% DMSO, 21% DMSO, 21.5% DMSO,22% DMSO, 22.5% DMSO, 23% DMSO, 23.5% DMSO, 24% DMSO, 24.5% DMSO, 25%DMSO, 25.5% DMSO, 26% DMSO, 26.5% DMSO, 27% DMSO, 27.5% DMSO, 28% DMSO,28.5% DMSO, 29% DMSO, 29.5% DMSO, 30% DMSO, 40% DMSO or less than 50%DMSO.

In some cases, the concentration of normosol in the cryopreservationmedium may be less than 0.1% normosol, 0.2% normosol, 0.3% normosol,0.4% normosol, 0.5% normosol, 0.6% normosol, 0.7% normosol, 0.8%normosol, 0.9% normosol, 1.0% normosol, 1.1% normosol, 1.2% normosol,1.3% normosol, 1.4% normosol, 1.5% normosol, 1.6% normosol, 1.7%normosol, 1.8% normosol, 1.9% normosol, 2.0% normosol, 2.1% normosol,2.2% normosol, 2.3% normosol, 2.4% normosol, 2.5% normosol, 2.6%normosol, 2.7% normosol, 2.8% normosol, 2.9% normosol, 3.0% normosol,3.1% normosol, 3.2% normosol, 3.3% normosol, 3.4% normosol, 3.5%normosol, 3.6% normosol, 3.7% normosol, 3.8% normosol, 3.9% normosol,4.0% normosol, 4.1% normosol, 4.2% normosol, 4.3% normosol, 4.4%normosol, 4.5% normosol, 4.6% normosol, 4.7% normosol, 4.8% normosol,4.9% normosol, 5.0% normosol, 5.1% normosol, 5.2% normosol, 5.3%normosol, 5.4% normosol, 5.5% normosol, 5.6% normosol, 5.7% normosol,5.8% normosol, 5.9% normosol, 6.0% normosol, 6.1% normosol, 6.2%normosol, 6.3% normosol, 6.4% normosol, 6.5% normosol, 6.6% normosol,6.7% normosol, 6.8% normosol, 6.9% normosol, 7.0% normosol, 7.1%normosol, 7.2% normosol, 7.3% normosol, 7.4% normosol, 7.5% normosol,7.6% normosol, 7.7% normosol, 7.8% normosol, 7.9% normosol, 8.0%normosol, 8.1% normosol, 8.2% normosol, 8.3% normosol, 8.4% normosol,8.5% normosol, 8.6% normosol, 8.7% normosol, 8.8% normosol, 8.9%normosol, 9.0% normosol, 9.1% normosol, 9.2% normosol, 9.3% normosol,9.4% normosol, 9.5% normosol, 9.6% normosol, 9.7% normosol, 9.8%normosol, 9.9% normosol, 10% normosol, 10.5% normosol, 11% normosol,11.5% normosol, 12% normosol, 12.5% normosol, 13% normosol, 13.5%normosol, 14% normosol, 14.5% normosol, 15% normosol, 15.5% normosol,16% normosol, 16.5% normosol, 17% normosol, 17.5% normosol, 18%normosol, 18.5% normosol, 19% normosol, 20% normosol, 20.5% normosol,21% normosol, 21.5% normosol, 22% normosol, 22.5% normosol, 23%normosol, 23.5% normosol, 24% normosol, 24.5% normosol, 25% normosol,25.5% normosol, 26% normosol, 26.5% normosol, 27% normosol, 27.5%normosol, 28% normosol, 28.5% normosol, 29% normosol, 29.5% normosol,30% normosol, 40% normosol or less than 50% normosol.

In some cases, the concentration of Hetastarch in the cryopreservationmedium may be less than 0.1% Hetastarch, 0.2% Hetastarch, 0.3%Hetastarch, 0.4% Hetastarch, 0.5% Hetastarch, 0.6% Hetastarch, 0.7%Hetastarch, 0.8% Hetastarch, 0.9% Hetastarch, 1.0% Hetastarch, 1.1%Hetastarch, 1.2% Hetastarch, 1.3% Hetastarch, 1.4% Hetastarch, 1.5%Hetastarch, 1.6% Hetastarch, 1.7% Hetastarch, 1.8% Hetastarch, 1.9%Hetastarch, 2.0% Hetastarch, 2.1% Hetastarch, 2.2% Hetastarch, 2.3%Hetastarch, 2.4% Hetastarch, 2.5% Hetastarch, 2.6% Hetastarch, 2.7%Hetastarch, 2.8% Hetastarch, 2.9% Hetastarch, 3.0% Hetastarch, 3.1%Hetastarch, 3.2% Hetastarch, 3.3% Hetastarch, 3.4% Hetastarch, 3.5%Hetastarch, 3.6% Hetastarch, 3.7% Hetastarch, 3.8% Hetastarch, 3.9%Hetastarch, 4.0% Hetastarch, 4.1% Hetastarch, 4.2% Hetastarch, 4.3%Hetastarch, 4.4% Hetastarch, 4.5% Hetastarch, 4.6% Hetastarch, 4.7%Hetastarch, 4.8% Hetastarch, 4.9% Hetastarch, 5.0% Hetastarch, 5.1%Hetastarch, 5.2% Hetastarch, 5.3% Hetastarch, 5.4% Hetastarch, 5.5%Hetastarch, 5.6% Hetastarch, 5.7% Hetastarch, 5.8% Hetastarch, 5.9%Hetastarch, 6.0% Hetastarch, 6.1% Hetastarch, 6.2% Hetastarch, 6.3%Hetastarch, 6.4% Hetastarch, 6.5% Hetastarch, 6.6% Hetastarch, 6.7%Hetastarch, 6.8% Hetastarch, 6.9% Hetastarch, 7.0% Hetastarch, 7.1%Hetastarch, 7.2% Hetastarch, 7.3% Hetastarch, 7.4% Hetastarch, 7.5%Hetastarch, 7.6% Hetastarch, 7.7% Hetastarch, 7.8% Hetastarch, 7.9%Hetastarch, 8.0% Hetastarch, 8.1% Hetastarch, 8.2% Hetastarch, 8.3%Hetastarch, 8.4% Hetastarch, 8.5% Hetastarch, 8.6% Hetastarch, 8.7%Hetastarch, 8.8% Hetastarch, 8.9% Hetastarch, 9.0% Hetastarch, 9.1%Hetastarch, 9.2% Hetastarch, 9.3% Hetastarch, 9.4% Hetastarch, 9.5%Hetastarch, 9.6% Hetastarch, 9.7% Hetastarch, 9.8% Hetastarch, 9.9%Hetastarch, 10% Hetastarch, 10.5% Hetastarch, 11% Hetastarch, 11.5%Hetastarch, 12% Hetastarch, 12.5% Hetastarch, 13% Hetastarch, 13.5%Hetastarch, 14% Hetastarch, 14.5% Hetastarch, 15% Hetastarch, 15.5%Hetastarch, 16% Hetastarch, 16.5% Hetastarch, 17% Hetastarch, 17.5%Hetastarch, 18% Hetastarch, 18.5% Hetastarch, 19% Hetastarch, 20%Hetastarch, 20.5% Hetastarch, 21% Hetastarch, 21.5% Hetastarch, 22%Hetastarch, 22.5% Hetastarch, 23% Hetastarch, 23.5% Hetastarch, 24%Hetastarch, 24.5% Hetastarch, 25% Hetastarch, 25.5% Hetastarch, 26%Hetastarch, 26.5% Hetastarch, 27% Hetastarch, 27.5% Hetastarch, 28%Hetastarch, 28.5% Hetastarch, 29% Hetastarch, 29.5% Hetastarch, 30%Hetastarch, 40% Hetastarch or less than 50% Hetastarch.

In some cases, the concentration of HSA in the cryopreservation mediummay be less than 0.1% HSA, 0.2% HSA, 0.3% HSA, 0.4% HSA, 0.5% HSA, 0.6%HSA, 0.7% HSA, 0.8% HSA, 0.9% HSA, 1.0% HSA, 1.1% HSA, 1.2% HSA, 1.3%HSA, 1.4% HSA, 1.5% HSA, 1.6% HSA, 1.7% HSA, 1.8% HSA, 1.9% HSA, 2.0%HSA, 2.1% HSA, 2.2% HSA, 2.3% HSA, 2.4% HSA, 2.5% HSA, 2.6% HSA, 2.7%HSA, 2.8% HSA, 2.9% HSA, 3.0% HSA, 3.1% HSA, 3.2% HSA, 3.3% HSA, 3.4%HSA, 3.5% HSA, 3.6% HSA, 3.7% HSA, 3.8% HSA, 3.9% HSA, 4.0% HSA, 4.1%HSA, 4.2% HSA, 4.3% HSA, 4.4% HSA, 4.5% HSA, 4.6% HSA, 4.7% HSA, 4.8%HSA, 4.9% HSA, 5.0% HSA, 5.1% HSA, 5.2% HSA, 5.3% HSA, 5.4% HSA, 5.5%HSA, 5.6% HSA, 5.7% HSA, 5.8% HSA, 5.9% HSA, 6.0% HSA, 6.1% HSA, 6.2%HSA, 6.3% HSA, 6.4% HSA, 6.5% HSA, 6.6% HSA, 6.7% HSA, 6.8% HSA, 6.9%HSA, 7.0% HSA, 7.1% HSA, 7.2% HSA, 7.3% HSA, 7.4% HSA, 7.5% HSA, 7.6%HSA, 7.7% HSA, 7.8% HSA, 7.9% HSA, 8.0% HSA, 8.1% HSA, 8.2% HSA, 8.3%HSA, 8.4% HSA, 8.5% HSA, 8.6% HSA, 8.7% HSA, 8.8% HSA, 8.9% HSA, 9.0%HSA, 9.1% HSA, 9.2% HSA, 9.3% HSA, 9.4% HSA, 9.5% HSA, 9.6% HSA, 9.7%HSA, 9.8% HSA, 9.9% HSA, 10% HSA, 10.5% HSA, 11% HSA, 11.5% HSA, 12%HSA, 12.5% HSA, 13% HSA, 13.5% HSA, 14% HSA, 14.5% HSA, 15% HSA, 15.5%HSA, 16% HSA, 16.5% HSA, 17% HSA, 17.5% HSA, 18% HSA, 18.5% HSA, 19%HSA, 20% HSA, 20.5% HSA, 21% HSA, 21.5% HSA, 22% HSA, 22.5% HSA, 23%HSA, 23.5% HSA, 24% HSA, 24.5% HSA, 25% HSA, 25.5% HSA, 26% HSA, 26.5%HSA, 27% HSA, 27.5% HSA, 28% HSA, 28.5% HSA, 29% HSA, 29.5% HSA, 30%HSA, 40% HSA or less than 50% HSA.

In some cases, the cryopreservation medium may contain other componentsin order to cryopreserve the hematopoietic cells in accordance with andfor use with the methods described herein.

For the methods described herein, hematopoietic cells can be frozen(e.g., cryopreserved) after isolation or after isolation andpurification from the solid organ donor. In some cases, hematopoieticcells may be cryopreserved using a cryopreservation medium and method ofcryopreservation known to those of skill in the art. In some cases, thehematopoietic cells may be cryopreserved using a cryopreservation mediumcontaining dimethylsulfoxide (DMSO), fetal calf serum (FCS) and RPMImedium.

In some cases, the concentration of DMSO in the cryopreservation mediummay be less than 0.1% DMSO, 0.2% DMSO, 0.3% DMSO, 0.4% DMSO, 0.5% DMSO,0.6% DMSO, 0.7% DMSO, 0.8% DMSO, 0.9% DMSO, 1.0% DMSO, 1.1% DMSO, 1.2%DMSO, 1.3% DMSO, 1.4% DMSO, 1.5% DMSO, 1.6% DMSO, 1.7% DMSO, 1.8% DMSO,1.9% DMSO, 2.0% DMSO, 2.1% DMSO, 2.2% DMSO, 2.3% DMSO, 2.4% DMSO, 2.5%DMSO, 2.6% DMSO, 2.7% DMSO, 2.8% DMSO, 2.9% DMSO, 3.0% DMSO, 3.1% DMSO,3.2% DMSO, 3.3% DMSO, 3.4% DMSO, 3.5% DMSO, 3.6% DMSO, 3.7% DMSO, 3.8%DMSO, 3.9% DMSO, 4.0% DMSO, 4.1% DMSO, 4.2% DMSO, 4.3% DMSO, 4.4% DMSO,4.5% DMSO, 4.6% DMSO, 4.7% DMSO, 4.8% DMSO, 4.9% DMSO, 5.0% DMSO, 5.1%DMSO, 5.2% DMSO, 5.3% DMSO, 5.4% DMSO, 5.5% DMSO, 5.6% DMSO, 5.7% DMSO,5.8% DMSO, 5.9% DMSO, 6.0% DMSO, 6.1% DMSO, 6.2% DMSO, 6.3% DMSO, 6.4%DMSO, 6.5% DMSO, 6.6% DMSO, 6.7% DMSO, 6.8% DMSO, 6.9% DMSO, 7.0% DMSO,7.1% DMSO, 7.2% DMSO, 7.3% DMSO, 7.4% DMSO, 7.5% DMSO, 7.6% DMSO, 7.7%DMSO, 7.8% DMSO, 7.9% DMSO, 8.0% DMSO, 8.1% DMSO, 8.2% DMSO, 8.3% DMSO,8.4% DMSO, 8.5% DMSO, 8.6% DMSO, 8.7% DMSO, 8.8% DMSO, 8.9% DMSO, 9.0%DMSO, 9.1% DMSO, 9.2% DMSO, 9.3% DMSO, 9.4% DMSO, 9.5% DMSO, 9.6% DMSO,9.7% DMSO, 9.8% DMSO, 9.9% DMSO, 10% DMSO, 10.5% DMSO, 11% DMSO, 11.5%DMSO, 12% DMSO, 12.5% DMSO, 13% DMSO, 13.5% DMSO, 14% DMSO, 14.5% DMSO,15% DMSO, 15.5% DMSO, 16% DMSO, 16.5% DMSO, 17% DMSO, 17.5% DMSO, 18%DMSO, 18.5% DMSO, 19% DMSO, 20% DMSO, 20.5% DMSO, 21% DMSO, 21.5% DMSO,22% DMSO, 22.5% DMSO, 23% DMSO, 23.5% DMSO, 24% DMSO, 24.5% DMSO, 25%DMSO, 25.5% DMSO, 26% DMSO, 26.5% DMSO, 27% DMSO, 27.5% DMSO, 28% DMSO,28.5% DMSO, 29% DMSO, 29.5% DMSO, 30% DMSO, 40% DMSO or less than 50%DMSO.

In some cases, the concentration of FCS in the cryopreservation mediummay be greater than 1.0% FCS, 2.0% FCS, 3.0% FCS, 4.0% FCS, 5.0% FCS,6.0% FCS, 7.0% FCS, 8.0% FCS, 9.0% FCS, 10% FCS, 10.5% FCS, 11% FCS,11.5% FCS, 12% FCS, 12.5% FCS, 13% FCS, 13.5% FCS, 14% FCS, 14.5% FCS,15% FCS, 15.5% FCS, 16% FCS, 16.5% FCS, 17% FCS, 17.5% FCS, 18% FCS,18.5% FCS, 19% FCS, 20% FCS, 20.5% FCS, 21% FCS, 21.5% FCS, 22% FCS,22.5% FCS, 23% FCS, 23.5% FCS, 24% FCS, 24.5% FCS, 25% FCS, 25.5% FCS,26% FCS, 26.5% FCS, 27% FCS, 27.5% FCS, 28% FCS, 28.5% FCS, 29% FCS,29.5% FCS, 30% FCS, 30.5% FCS, 31% FCS, 31.5% FCS, 32% FCS, 32.5% FCS,33% FCS, 33.5% FCS, 34% FCS, 34.5% FCS, 35% FCS, 35.5% FCS, 36% FCS,36.5% FCS, 37% FCS, 37.5% FCS, 38% FCS, 38.5% FCS, 39% FCS, 40% FCS,40.5% FCS, 41% FCS, 41.5% FCS, 42% FCS, 42.5% FCS, 43% FCS, 43.5% FCS,44% FCS, 44.5% FCS, 45% FCS, 45.5% FCS, 46% FCS, 46.5% FCS, 47% FCS,47.5% FCS, 48% FCS, 48.5% FCS, 49% FCS, 50% FCS, 50.5% FCS, 51% FCS,51.5% FCS, 52% FCS, 52.5% FCS, 53% FCS, 53.5% FCS, 54% FCS, 54.5% FCS,55% FCS, 55.5% FCS, 56% FCS, 56.5% FCS, 57% FCS, 57.5% FCS, 58% FCS,58.5% FCS, 59% FCS, 60% FCS, 60.5% FCS, 61% FCS, 61.5% FCS, 62% FCS,62.5% FCS, 63% FCS, 63.5% FCS, 64% FCS, 64.5% FCS, 65% FCS, 65.5% FCS,66% FCS, 66.5% FCS, 67% FCS, 67.5% FCS, 68% FCS, 68.5% FCS, 69% FCS, 70%FCS, 70.5% FCS, 71% FCS, 71.5% FCS, 72% FCS, 72.5% FCS, 73% FCS, 73.5%FCS, 74% FCS, 74.5% FCS, 75% FCS, 75.5% FCS, 76% FCS, 76.5% FCS, 77%FCS, 77.5% FCS, 78% FCS, 78.5% FCS, 79% FCS or greater than 80% FCS.

In some cases, the concentration of RPMI in the cryopreservation mediummay be greater than 1.0% RPMI, 2.0% RPMI, 3.0% RPMI, 4.0% RPMI, 5.0%RPMI, 6.0% RPMI, 7.0% RPMI, 8.0% RPMI, 9.0% RPMI, 10% RPMI, 10.5% RPMI,11% RPMI, 11.5% RPMI, 12% RPMI, 12.5% RPMI, 13% RPMI, 13.5% RPMI, 14%RPMI, 14.5% RPMI, 15% RPMI, 15.5% RPMI, 16% RPMI, 16.5% RPMI, 17% RPMI,17.5% RPMI, 18% RPMI, 18.5% RPMI, 19% RPMI, 20% RPMI, 20.5% RPMI, 21%RPMI, 21.5% RPMI, 22% RPMI, 22.5% RPMI, 23% RPMI, 23.5% RPMI, 24% RPMI,24.5% RPMI, 25% RPMI, 25.5% RPMI, 26% RPMI, 26.5% RPMI, 27% RPMI, 27.5%RPMI, 28% RPMI, 28.5% RPMI, 29% RPMI, 29.5% RPMI, 30% RPMI, 30.5% RPMI,31% RPMI, 31.5% RPMI, 32% RPMI, 32.5% RPMI, 33% RPMI, 33.5% RPMI, 34%RPMI, 34.5% RPMI, 35% RPMI, 35.5% RPMI, 36% RPMI, 36.5% RPMI, 37% RPMI,37.5% RPMI, 38% RPMI, 38.5% RPMI, 39% RPMI, 40% RPMI, 40.5% RPMI, 41%RPMI, 41.5% RPMI, 42% RPMI, 42.5% RPMI, 43% RPMI, 43.5% RPMI, 44% RPMI,44.5% RPMI, 45% RPMI, 45.5% RPMI, 46% RPMI, 46.5% RPMI, 47% RPMI, 47.5%RPMI, 48% RPMI, 48.5% RPMI, 49% RPMI, 50% RPMI, 50.5% RPMI, 51% RPMI,51.5% RPMI, 52% RPMI, 52.5% RPMI, 53% RPMI, 53.5% RPMI, 54% RPMI, 54.5%RPMI, 55% RPMI, 55.5% RPMI, 56% RPMI, 56.5% RPMI, 57% RPMI, 57.5% RPMI,58% RPMI, 58.5% RPMI, 59% RPMI, 60% RPMI, 60.5% RPMI, 61% RPMI, 61.5%RPMI, 62% RPMI, 62.5% RPMI, 63% RPMI, 63.5% RPMI, 64% RPMI, 64.5% RPMI,65% RPMI, 65.5% RPMI, 66% RPMI, 66.5% RPMI, 67% RPMI, 67.5% RPMI, 68%RPMI, 68.5% RPMI, 69% RPMI, 70% RPMI, 70.5% RPMI, 71% RPMI, 71.5% RPMI,72% RPMI, 72.5% RPMI, 73% RPMI, 73.5% RPMI, 74% RPMI, 74.5% RPMI, 75%RPMI, 75.5% RPMI, 76% RPMI, 76.5% RPMI, 77% RPMI, 77.5% RPMI, 78% RPMI,78.5% RPMI, 79% RPMI or greater than 80% RPMI.

In some cases, the cryopreservation medium may contain other componentsin order to cryopreserve the hematopoietic cells in accordance with andfor use with the methods described herein.

Cryopreservation of hematopoietic cells includes a process of controlledrate freezing the cells once contained within cryopreservation medium.In some cases, a cryofreezer equipped with a computer to control therate and temperatures of controlled rate freezing can be used to performcryopreservation of the hematopoietic cells. For example, thehematopoietic cells may be placed in a cryofreezer with a chambertemperature at or below 6.5° C. The computer may control the rate andtemperatures of controlled rate freezing such that the cryofreezerreaches a temperature of at least or below −130° C. such that thehematopoietic cells are preserved in manner in accordance with themethods described herein. In some cases, the cryofreezer uses liquidnitrogen to control the temperature of the freezer at which thehematopoietic cells are stored.

In some cases, the hematopoietic cells may be cryopreserved and storedin a cryofreezer prior to delivery to the recipient. In some cases, thehematopoietic cells may be cryopreserved for less than one day, twodays, three days, four days, five days, six days, seven days, eightdays, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days,16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days,24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days,32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days,40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days,48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days,56 days, 57 days, 58 days, 59 days or less than 60 days.

In some cases, the hematopoietic cells may be cryopreserved for lessthan one month, two months, three months, four months, five months, sixmonths, seven months, eight months, nine months, ten months, 11 months,12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24months, 25 months, 26 months, 27 months, 28 months, 29 months, 30months, 31 months, 32 months, 33 months, 34 months, 35 months, 36months, 37 months, 38 months, 39 months, 40 months, 41 months, 42months, 43 months, 44 months, 45 months, 46 months, 47 months, 48months, 49 months, 50 months, 51 months, 52 months, 53 months, 54months, 55 months, 56 months, 57 months, 58 months, 59 months or lessthan 60 months.

In some cases, the hematopoietic cells may be cryopreserved for lessthan one year, two years, three years, four years, five years, sixyears, seven years, eight years, nine years, ten years, 11 years, 12years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47years, 48 years, 49 years, 50 years, 51 years, 52 years, 53 years, 54years, 55 years, 56 years, 57 years, 58 years, 59 years or less than 60years.

In some cases, the hematopoietic cells may be cryopreserved for morethan one day, two days, three days, four days, five days, six days,seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days,14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days,22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days,30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days,38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days,46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days,54 days, 55 days, 56 days, 57 days, 58 days, 59 days or more 60 days.

In some cases, the hematopoietic cells may be cryopreserved for morethan one month, two months, three months, four months, five months, sixmonths, seven months, eight months, nine months, ten months, 11 months,12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24months, 25 months, 26 months, 27 months, 28 months, 29 months, 30months, 31 months, 32 months, 33 months, 34 months, 35 months, 36months, 37 months, 38 months, 39 months, 40 months, 41 months, 42months, 43 months, 44 months, 45 months, 46 months, 47 months, 48months, 49 months, 50 months, 51 months, 52 months, 53 months, 54months, 55 months, 56 months, 57 months, 58 months, 59 months or formore than 60 months.

In some cases, the hematopoietic cells may be cryopreserved for morethan one year, two years, three years, four years, five years, sixyears, seven years, eight years, nine years, ten years, 11 years, 12years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47years, 48 years, 49 years, 50 years, 51 years, 52 years, 53 years, 54years, 55 years, 56 years, 57 years, 58 years, 59 years or more than 60years.

In some cases, cryopreservation may result in hematopoietic cell deathwhich is determined upon thawing of the cells prior to infusion into therecipient. Using conventional methods of determining cell death (e.g.,trypan blue staining, flow cytometry, etc.) known to those of skill inthe art, the percent of dead cells in batch of cryopreservedhematopoietic cells may be determined. In some cases, after thawingcryopreserved cells, there may be less than 0.1% dead cells, 0.2% deadcells, 0.3% dead cells, 0.4% dead cells, 0.5% dead cells, 0.6% deadcells, 0.7% dead cells, 0.8% dead cells, 0.9% dead cells, 1.0% deadcells, 1.1% dead cells, 1.2% dead cells, 1.3% dead cells, 1.4% deadcells, 1.5% dead cells, 1.6% dead cells, 1.7% dead cells, 1.8% deadcells, 1.9% dead cells, 2.0% dead cells, 2.1% dead cells, 2.2% deadcells, 2.3% dead cells, 2.4% dead cells, 2.5% dead cells, 2.6% deadcells, 2.7% dead cells, 2.8% dead cells, 2.9% dead cells, 3.0% deadcells, 3.1% dead cells, 3.2% dead cells, 3.3% dead cells, 3.4% deadcells, 3.5% dead cells, 3.6% dead cells, 3.7% dead cells, 3.8% deadcells, 3.9% dead cells, 4.0% dead cells, 4.1% dead cells, 4.2% deadcells, 4.3% dead cells, 4.4% dead cells, 4.5% dead cells, 4.6% deadcells, 4.7% dead cells, 4.8% dead cells, 4.9% dead cells, 5.0% deadcells, 5.1% dead cells, 5.2% dead cells, 5.3% dead cells, 5.4% deadcells, 5.5% dead cells, 5.6% dead cells, 5.7% dead cells, 5.8% deadcells, 5.9% dead cells, 6.0% dead cells, 6.1% dead cells, 6.2% deadcells, 6.3% dead cells, 6.4% dead cells, 6.5% dead cells, 6.6% deadcells, 6.7% dead cells, 6.8% dead cells, 6.9% dead cells, 7.0% deadcells, 7.1% dead cells, 7.2% dead cells, 7.3% dead cells, 7.4% deadcells, 7.5% dead cells, 7.6% dead cells, 7.7% dead cells, 7.8% deadcells, 7.9% dead cells, 8.0% dead cells, 8.1% dead cells, 8.2% deadcells, 8.3% dead cells, 8.4% dead cells, 8.5% dead cells, 8.6% deadcells, 8.7% dead cells, 8.8% dead cells, 8.9% dead cells, 9.0% deadcells, 9.1% dead cells, 9.2% dead cells, 9.3% dead cells, 9.4% deadcells, 9.5% dead cells, 9.6% dead cells, 9.7% dead cells, 9.8% deadcells, 9.9% dead cells, 10% dead cells, 10.5% dead cells, 11% deadcells, 11.5% dead cells, 12% dead cells, 12.5% dead cells, 13% deadcells, 13.5% dead cells, 14% dead cells, 14.5% dead cells, 15% deadcells, 15.5% dead cells, 16% dead cells, 16.5% dead cells, 17% deadcells, 17.5% dead cells, 18% dead cells, 18.5% dead cells, 19% deadcells, 20% dead cells, 20.5% dead cells, 21% dead cells, 21.5% deadcells, 22% dead cells, 22.5% dead cells, 23% dead cells, 23.5% deadcells, 24% dead cells, 24.5% dead cells, 25% dead cells, 25.5% deadcells, 26% dead cells, 26.5% dead cells, 27% dead cells, 27.5% deadcells, 28% dead cells, 28.5% dead cells, 29% dead cells, 29.5% deadcells, 30% dead cells, 40% dead cells or less than 50% dead cells.

Isolation and Purification of Hematopoietic Stem Cells

For the methods described herein, hematopoietic stem cells may bederived from bone marrow, peripheral blood, or umbilical cord blood. Insome cases, the hematopoietic stem cells may be HLA-matched between thedonor and the recipient. In other cases, the hematopoietic stem cellsmay be HLA-mismatched between the donor and the recipient.

In some cases, specific types of cells may be isolated and purified fromthe hematopoietic stem cells. In some cases, the cells that may beisolated and purified from the hematopoietic stem cells are CD34+ cellsand CD3+ cells. In some cases, the CD34+ and CD3+ cells are isolatedfrom the same fraction of hematopoietic stem cells. In some cases, theCD34+ and CD3+ cells are isolated from a different fraction ofhematopoietic stem cells. In some cases, the CD34+ cells are progenitorcells. In some cases, the CD3+ cells are T cells.

In some cases, CD34+ cells are isolated and purified from the donorhematopoietic cells. For example, CD34+ cells may be isolated andpurified from the donor hematopoietic cells by selectively binding asuitable CD34 affinity reagent. In some cases, a CD34 affinity reagentmay be an antibody, a full-length antibody, a fragment of an antibody, anaturally occurring antibody, a synthetic antibody, an engineeredantibody, a full-length affibody, a fragment of an affibody, afull-length affilin, a fragment of an affilin, a full-length anticalin,a fragment of an anticalin, a full-length avimer, a fragment of anavimer, a full-length DARPin, a fragment of a DARPin, a full-lengthfynomer, a fragment of a fynomer, a full-length kunitz domain peptide, afragment of a kunitz domain peptide, a full-length monobody, a fragmentof a monobody, a peptide, a polyaminoacid, or the like.

In some cases, the affinity reagent is directly conjugated to adetection reagent and/or purification reagent. In some cases, thedetection reagent and purification reagent are the same. In other cases,the detection reagent and purification reagent are different. Forexample, the detection reagent and/or purification reagent isfluorescent, magnetic, or the like. In some cases, the detection reagentand/or purification reagent is a magnetic particle for columnpurification. For example, magnetic column purification may be performedusing the Miltenyi system of columns, antibodies, buffers, preparationmaterials and reagents, etc. known to those of skill in the art.

In some cases, CD34+ cells isolated and purified using a magneticparticle may contain iron. The iron content of isolated and purifiedCD34+ cells may be greater after isolation and purification usingmagnetic particles than the iron content in the CD34+ cells prior toisolation and purification. For example, isolated and purified CD34+cells may contain less than 500 pg of iron/cell, 450 pg of iron/cell,400 pg of iron/cell, 350 pg of iron/cell, 300 pg of iron/cell, 250 pg ofiron/cell, 225 pg of iron/cell, 200 pg of iron/cell, 190 pg ofiron/cell, 180 pg of iron/cell, 170 pg of iron/cell, 160 pg ofiron/cell, 150 pg of iron/cell, 140 pg of iron/cell, 130 pg ofiron/cell, 120 pg of iron/cell, 110 pg of iron/cell, 109 pg ofiron/cell, 108 pg of iron/cell, 107 pg of iron/cell, 106 pg ofiron/cell, 105 pg of iron/cell, 104 pg of iron/cell, 103 pg ofiron/cell, 102 pg of iron/cell, 101 pg of iron/cell, 100 pg ofiron/cell, 99 pg of iron/cell, 98 pg of iron/cell, 97 pg of iron/cell,96 pg of iron/cell, 95 pg of iron/cell, 94 pg of iron/cell, 93 pg ofiron/cell, 92 pg of iron/cell, 91 pg of iron/cell, 90 pg of iron/cell,89 pg of iron/cell, 88 pg of iron/cell, 87 pg of iron/cell, 86 pg ofiron/cell, 85 pg of iron/cell, 84 pg of iron/cell, 83 pg of iron/cell,82 pg of iron/cell, 81 pg of iron/cell, 80 pg of iron/cell, 79 pg ofiron/cell, 78 pg of iron/cell, 77 pg of iron/cell, 76 pg of iron/cell,75 pg of iron/cell, 74 pg of iron/cell, 73 pg of iron/cell, 72 pg ofiron/cell, 71 pg of iron/cell, 70 pg of iron/cell, 69 pg of iron/cell,68 pg of iron/cell, 67 pg of iron/cell, 66 pg of iron/cell, 65 pg ofiron/cell, 64 pg of iron/cell, 63 pg of iron/cell, 62 pg of iron/cell,61 pg of iron/cell, 60 pg of iron/cell, 59 pg of iron/cell, 58 pg ofiron/cell, 57 pg of iron/cell, 56 pg of iron/cell, 55 pg of iron/cell,54 pg of iron/cell, 53 pg of iron/cell, 52 pg of iron/cell, 51 pg ofiron/cell, 50 pg of iron/cell, 49 pg of iron/cell, 48 pg of iron/cell,47 pg of iron/cell, 46 pg of iron/cell, 45 pg of iron/cell, 44 pg ofiron/cell, 43 pg of iron/cell, 42 pg of iron/cell, 41 pg of iron/cell,40 pg of iron/cell, 39 pg of iron/cell, 38 pg of iron/cell, 37 pg ofiron/cell, 36 pg of iron/cell, 35 pg of iron/cell, 34 pg of iron/cell,33 pg of iron/cell, 32 pg of iron/cell, 31 pg of iron/cell, 30 pg ofiron/cell, 29 pg of iron/cell, 28 pg of iron/cell, 27 pg of iron/cell,26 pg of iron/cell, 25 pg of iron/cell, 24 pg of iron/cell, 23 pg ofiron/cell, 22 pg of iron/cell, 21 pg of iron/cell, 20 pg of iron/cell,19 pg of iron/cell, 18 pg of iron/cell, 17 pg of iron/cell, 16 pg ofiron/cell, 15 pg of iron/cell, 14 pg of iron/cell, 13 pg of iron/cell,12 pg of iron/cell, 11 pg of iron/cell, 10 pg of iron/cell, 9 pg ofiron/cell, 8 pg of iron/cell, 7 pg of iron/cell, 6 pg of iron/cell, 5 pgof iron/cell, 4 pg of iron/cell, 3 pg of iron/cell, 2 pg of iron/cell,or less than 1 pg of iron/cell.

In some cases, CD3+ cells are isolated and purified from the donorhematopoietic cells. For example, CD3+ cells may be isolated andpurified from the donor hematopoietic cells by selectively binding asuitable CD3 affinity reagent. In some cases, a CD3 affinity reagent maybe an antibody, a full-length antibody, a fragment of an antibody, anaturally occurring antibody, a synthetic antibody, an engineeredantibody, a full-length affibody, a fragment of an affibody, afull-length affilin, a fragment of an affilin, a full-length anticalin,a fragment of an anticalin, a full-length avimer, a fragment of anavimer, a full-length DARPin, a fragment of a DARPin, a full-lengthfynomer, a fragment of a fynomer, a full-length kunitz domain peptide, afragment of a kunitz domain peptide, a full-length monobody, a fragmentof a monobody, a peptide, a polyaminoacid, or the like.

In some cases, the affinity reagent is directly conjugated to adetection reagent and/or purification reagent. In some cases, thedetection reagent and purification reagent are the same. In other cases,the detection reagent and purification reagent are different. Forexample, the detection reagent and/or purification reagent isfluorescent, magnetic, or the like. In some cases, the detection reagentand/or purification reagent is a magnetic particle for columnpurification. For example, magnetic column purification may be performedusing the Miltenyi system of columns, antibodies, buffers, preparationmaterials and reagents, etc. known to those of skill in the art.

The CD3+ cells may be selected from one of two cell populations. In somecases, the apheresis product may be split into two portions, one portionused to isolate and purify CD3+ cells and the other portion to isolateand purify CD34+ cells. In some cases, CD34+ cells are isolated andpurified from the apheresis product creating a CD34-negative cellfraction. In some cases, the number of CD3+ cells in the CD34-negativefraction can be determined and a volume of the CD34-negative fractioncontaining an appropriate dose of CD3 cells combined with a volume ofisolated and purified CD34+ cells. For example, CD34+ cells and the CD3+cells are isolated from the at least one apheresis product using atleast an affinity agent and a column, the CD34+ cells located in aneluate from the column and the CD3+ cells located in a CD34-depletedflow-through fraction from the column. In another example, theCD34-depleted fraction of the apheresis product is the CD34-depletedflow-through fraction from an affinity column.

In some cases, the number of CD3+ cells in the negative fraction can bedetermined using an affinity reagent for CD3+. For example, an affinityreagent may be an antibody, a peptide or the like previously describedin the disclosure. In some cases, the affinity reagent may include adetection moiety. For example, a detection moiety may be fluorescent,magnetic or the like previously described in the disclosure.

In some cases, CD3+ cells isolated and purified using a magneticparticle may contain iron. The iron content of isolated and purifiedCD3+ cells may be greater after isolation and purification usingmagnetic particles than the iron content in the CD3+ cells prior toisolation and purification. For example, isolated and purified CD3+cells may contain less than 500 pg of iron/cell, 450 pg of iron/cell,400 pg of iron/cell, 350 pg of iron/cell, 300 pg of iron/cell, 250 pg ofiron/cell, 225 pg of iron/cell, 200 pg of iron/cell, 190 pg ofiron/cell, 180 pg of iron/cell, 170 pg of iron/cell, 160 pg ofiron/cell, 150 pg of iron/cell, 140 pg of iron/cell, 130 pg ofiron/cell, 120 pg of iron/cell, 110 pg of iron/cell, 109 pg ofiron/cell, 108 pg of iron/cell, 107 pg of iron/cell, 106 pg ofiron/cell, 105 pg of iron/cell, 104 pg of iron/cell, 103 pg ofiron/cell, 102 pg of iron/cell, 101 pg of iron/cell, 100 pg ofiron/cell, 99 pg of iron/cell, 98 pg of iron/cell, 97 pg of iron/cell,96 pg of iron/cell, 95 pg of iron/cell, 94 pg of iron/cell, 93 pg ofiron/cell, 92 pg of iron/cell, 91 pg of iron/cell, 90 pg of iron/cell,89 pg of iron/cell, 88 pg of iron/cell, 87 pg of iron/cell, 86 pg ofiron/cell, 85 pg of iron/cell, 84 pg of iron/cell, 83 pg of iron/cell,82 pg of iron/cell, 81 pg of iron/cell, 80 pg of iron/cell, 79 pg ofiron/cell, 78 pg of iron/cell, 77 pg of iron/cell, 76 pg of iron/cell,75 pg of iron/cell, 74 pg of iron/cell, 73 pg of iron/cell, 72 pg ofiron/cell, 71 pg of iron/cell, 70 pg of iron/cell, 69 pg of iron/cell,68 pg of iron/cell, 67 pg of iron/cell, 66 pg of iron/cell, 65 pg ofiron/cell, 64 pg of iron/cell, 63 pg of iron/cell, 62 pg of iron/cell,61 pg of iron/cell, 60 pg of iron/cell, 59 pg of iron/cell, 58 pg ofiron/cell, 57 pg of iron/cell, 56 pg of iron/cell, 55 pg of iron/cell,54 pg of iron/cell, 53 pg of iron/cell, 52 pg of iron/cell, 51 pg ofiron/cell, 50 pg of iron/cell, 49 pg of iron/cell, 48 pg of iron/cell,47 pg of iron/cell, 46 pg of iron/cell, 45 pg of iron/cell, 44 pg ofiron/cell, 43 pg of iron/cell, 42 pg of iron/cell, 41 pg of iron/cell,40 pg of iron/cell, 39 pg of iron/cell, 38 pg of iron/cell, 37 pg ofiron/cell, 36 pg of iron/cell, 35 pg of iron/cell, 34 pg of iron/cell,33 pg of iron/cell, 32 pg of iron/cell, 31 pg of iron/cell, 30 pg ofiron/cell, 29 pg of iron/cell, 28 pg of iron/cell, 27 pg of iron/cell,26 pg of iron/cell, 25 pg of iron/cell, 24 pg of iron/cell, 23 pg ofiron/cell, 22 pg of iron/cell, 21 pg of iron/cell, 20 pg of iron/cell,19 pg of iron/cell, 18 pg of iron/cell, 17 pg of iron/cell, 16 pg ofiron/cell, 15 pg of iron/cell, 14 pg of iron/cell, 13 pg of iron/cell,12 pg of iron/cell, 11 pg of iron/cell, 10 pg of iron/cell, 9 pg ofiron/cell, 8 pg of iron/cell, 7 pg of iron/cell, 6 pg of iron/cell, 5 pgof iron/cell, 4 pg of iron/cell, 3 pg of iron/cell, 2 pg of iron/cell,or less than 1 pg of iron/cell.

In some cases, both of the CD34+ and CD3+ cells isolated and purifiedusing a magnetic particle may contain iron. The iron content of isolatedand purified CD34+ and CD3+ cells may be greater after isolation andpurification using magnetic particles than the iron content in the CD34+and CD3+ cells prior to isolation and purification. For example,isolated and purified CD34+ and CD3+ cells may contain less than 500 pgof iron/cell, 450 pg of iron/cell, 400 pg of iron/cell, 350 pg ofiron/cell, 300 pg of iron/cell, 250 pg of iron/cell, 225 pg ofiron/cell, 200 pg of iron/cell, 190 pg of iron/cell, 180 pg ofiron/cell, 170 pg of iron/cell, 160 pg of iron/cell, 150 pg ofiron/cell, 140 pg of iron/cell, 130 pg of iron/cell, 120 pg ofiron/cell, 110 pg of iron/cell, 109 pg of iron/cell, 108 pg ofiron/cell, 107 pg of iron/cell, 106 pg of iron/cell, 105 pg ofiron/cell, 104 pg of iron/cell, 103 pg of iron/cell, 102 pg ofiron/cell, 101 pg of iron/cell, 100 pg of iron/cell, 99 pg of iron/cell,98 pg of iron/cell, 97 pg of iron/cell, 96 pg of iron/cell, 95 pg ofiron/cell, 94 pg of iron/cell, 93 pg of iron/cell, 92 pg of iron/cell,91 pg of iron/cell, 90 pg of iron/cell, 89 pg of iron/cell, 88 pg ofiron/cell, 87 pg of iron/cell, 86 pg of iron/cell, 85 pg of iron/cell,84 pg of iron/cell, 83 pg of iron/cell, 82 pg of iron/cell, 81 pg ofiron/cell, 80 pg of iron/cell, 79 pg of iron/cell, 78 pg of iron/cell,77 pg of iron/cell, 76 pg of iron/cell, 75 pg of iron/cell, 74 pg ofiron/cell, 73 pg of iron/cell, 72 pg of iron/cell, 71 pg of iron/cell,70 pg of iron/cell, 69 pg of iron/cell, 68 pg of iron/cell, 67 pg ofiron/cell, 66 pg of iron/cell, 65 pg of iron/cell, 64 pg of iron/cell,63 pg of iron/cell, 62 pg of iron/cell, 61 pg of iron/cell, 60 pg ofiron/cell, 59 pg of iron/cell, 58 pg of iron/cell, 57 pg of iron/cell,56 pg of iron/cell, 55 pg of iron/cell, 54 pg of iron/cell, 53 pg ofiron/cell, 52 pg of iron/cell, 51 pg of iron/cell, 50 pg of iron/cell,49 pg of iron/cell, 48 pg of iron/cell, 47 pg of iron/cell, 46 pg ofiron/cell, 45 pg of iron/cell, 44 pg of iron/cell, 43 pg of iron/cell,42 pg of iron/cell, 41 pg of iron/cell, 40 pg of iron/cell, 39 pg ofiron/cell, 38 pg of iron/cell, 37 pg of iron/cell, 36 pg of iron/cell,35 pg of iron/cell, 34 pg of iron/cell, 33 pg of iron/cell, 32 pg ofiron/cell, 31 pg of iron/cell, 30 pg of iron/cell, 29 pg of iron/cell,28 pg of iron/cell, 27 pg of iron/cell, 26 pg of iron/cell, 25 pg ofiron/cell, 24 pg of iron/cell, 23 pg of iron/cell, 22 pg of iron/cell,21 pg of iron/cell, 20 pg of iron/cell, 19 pg of iron/cell, 18 pg ofiron/cell, 17 pg of iron/cell, 16 pg of iron/cell, 15 pg of iron/cell,14 pg of iron/cell, 13 pg of iron/cell, 12 pg of iron/cell, 11 pg ofiron/cell, 10 pg of iron/cell, 9 pg of iron/cell, 8 pg of iron/cell, 7pg of iron/cell, 6 pg of iron/cell, 5 pg of iron/cell, 4 pg ofiron/cell, 3 pg of iron/cell, 2 pg of iron/cell, or less than 1 pg ofiron/cell.

Engineering and Preparing Hematopoietic Stem Cells for PharmaceuticalCompositions

A combination of CD34+ and CD3+ cells derived from the donor using themethods described herein may be engineered into a pharmaceuticalcomposition for administration to the solid organ recipient. In somecases, the hematopoietic cells may be engineered into a singlepharmaceutical composition for infusion into a recipient. In othercases, the hematopoietic cells may be engineered into multiplepharmaceutical compositions for infusion into a recipient. In somecases, the CD34+ and CD3+ cells may be HLA-matched between the donor andthe recipient. In other cases, the CD34+ and CD3+ cells may beHLA-mismatched between the donor and the recipient.

In some cases, the hematopoietic cells may be engineered into apharmaceutical composition having a pre-determined purity of CD34+hematopoietic cells. For example, the purity of the CD34+ progenitorcells in the engineered hematopoietic cells may be ≥30% purity, ≥40%purity, ≥50% purity, ≥55% purity, ≥60% purity, ≥65% purity, ≥70% purity,≥75% purity, ≥80% purity, ≥85% purity, ≥90% purity, ≥95% purity or ≥98%purity. In an another example, the purity of the CD34+ progenitor cellsin the engineered hematopoietic cells may be between 10 and 30% purity,15 and 35% purity, 20 and 40% purity, 25 and 45% purity, 30 and 50%purity, 35 and 55% purity, 40 and 60% purity, 45 and 65% purity, 50 and70% purity, 55 and 75% purity, 60 and 80% purity, 65 and 85% purity, 70and 90% purity, 75 and 95% purity, and 80 and 100% purity. In anexemplary case, the purity of the CD34+ progenitor cells in theengineered hematopoietic cells is ≥70% purity.

For example, the purity of the CD3+ cells in the engineeredhematopoietic cells may be ≥30% purity, ≥40% purity, ≥50% purity, ≥55%purity, ≥60% purity, ≥65% purity, ≥70% purity, ≥75% purity, ≥80% purity,≥85% purity, ≥90% purity, ≥95% purity or ≥98% purity. In an anotherexample, the purity of the CD3+ cells in the engineered hematopoieticcells may be between 10 and 30% purity, 15 and 35% purity, 20 and 40%purity, 25 and 45% purity, 30 and 50% purity, 35 and 55% purity, 40 and60% purity, 45 and 65% purity, 50 and 70% purity, 55 and 75% purity, 60and 80% purity, 65 and 85% purity, 70 and 90% purity, 75 and 95% purity,and 80 and 100% purity. In an exemplary case, the purity of the CD3+cells in the engineered hematopoietic cells is ≥70% purity prior tocombining with the CD34+ cells.

In some cases, the hematopoietic cells may be engineered into a producthaving a pre-determined purity of CD34+ progenitor cells. For example,the purity of the CD34+ progenitor cells in the engineered hematopoieticcells may be ≥30% purity, ≥40% purity, ≥50% purity, ≥55% purity, ≥60%purity, ≥65% purity, ≥70% purity, ≥75% purity, ≥80% purity, ≥85% purity,≥90% purity, ≥95% purity or ≥98% purity. In an another example, thepurity of the CD34+ progenitor cells in the engineered hematopoieticcells may be between 10 and 30% purity, 15 and 35% purity, 20 and 40%purity, 25 and 45% purity, 30 and 50% purity, 35 and 55% purity, 40 and60% purity, 45 and 65% purity, 50 and 70% purity, 55 and 75% purity, 60and 80% purity, 65 and 85% purity, 70 and 90% purity, 75 and 95% purity,and 80 and 100% purity. In an exemplary case, the purity of the CD34+progenitor cells in the engineered hematopoietic cells is ≥70% purityprior to combining with the CD3+ cells.

The engineered hematopoietic cells may contain a specific number ofCD34+ cells for injection into a recipient. In some cases, the targetdose of CD34+ cells to be injected into a recipient is ≥10×10³ cells/kgof body weight, ≥15×10³ cells/kg of body weight, ≥20×10³ cells/kg ofbody weight, ≥25×10³ cells/kg of body weight, ≥30×10³ cells/kg of bodyweight, ≥35×10³ cells/kg of body weight, ≥40×10³ cells/kg of bodyweight, ≥45×10³ cells/kg of body weight, ≥50×10³ cells/kg of bodyweight, ≥55×10³ cells/kg of body weight, ≥60×10³ cells/kg of bodyweight, ≥65×10³ cells/kg of body weight, ≥70×10³ cells/kg of bodyweight, ≥75×10³ cells/kg of body weight, ≥80×10³ cells/kg of bodyweight, ≥85×10³ cells/kg of body weight, 90×10³ cells/kg of body weight,≥95×10³ cells/kg of body weight, ≥10×10⁴ cells/kg of body weight,≥15×10⁴ cells/kg of body weight, ≥20×10⁴ cells/kg of body weight,≥25×10⁴ cells/kg of body weight, ≥30×10⁴ cells/kg of body weight,≥35×10⁴ cells/kg of body weight, ≥40×10⁴ cells/kg of body weight,≥45×10⁴ cells/kg of body weight, ≥50×10⁴ cells/kg of body weight,≥55×10⁴ cells/kg of body weight, ≥60×10⁴ cells/kg of body weight,≥65×10⁴ cells/kg of body weight, ≥70×10⁴ cells/kg of body weight,≥75×10⁴ cells/kg of body weight, ≥80×10⁴ cells/kg of body weight,≥85×10⁴ cells/kg of body weight, 90×10⁴ cells/kg of body weight, ≥95×10⁴cells/kg of body weight, ≥10×10⁵ cells/kg of body weight, ≥15×10⁵cells/kg of body weight, ≥20×10⁵ cells/kg of body weight, ≥25×10⁵cells/kg of body weight, ≥30×10⁵ cells/kg of body weight, ≥35×10⁵cells/kg of body weight, ≥40×10⁵ cells/kg of body weight, ≥45×10⁵cells/kg of body weight, ≥50×10⁵ cells/kg of body weight, ≥55×10⁵cells/kg of body weight, ≥60×10⁵ cells/kg of body weight, ≥65×10⁵cells/kg of body weight, ≥70×10⁵ cells/kg of body weight, ≥75×10⁵cells/kg of body weight, ≥80×10⁵ cells/kg of body weight, ≥85×10⁵cells/kg of body weight, 90×10⁵ cells/kg of body weight, ≥95×10⁵cells/kg of body weight, ≥10×10⁶ cells/kg of body weight, ≥15×10⁶cells/kg of body weight, ≥20×10⁶ cells/kg of body weight, ≥25×10⁶cells/kg of body weight, ≥30×10⁶ cells/kg of body weight, ≥35×10⁶cells/kg of body weight, ≥40×10⁶ cells/kg of body weight, ≥45×10⁶cells/kg of body weight, ≥50×10⁶ cells/kg of body weight, ≥55×10⁶cells/kg of body weight, ≥60×10⁶ cells/kg of body weight, ≥65×10⁶cells/kg of body weight, ≥70×10⁶ cells/kg of body weight, ≥75×10⁶cells/kg of body weight, ≥80×10⁶ cells/kg of body weight, ≥85×10⁶cells/kg of body weight, 90×10⁶ cells/kg of body weight, ≥95×10⁶cells/kg of body weight, ≥10×10⁷ cells/kg of body weight, ≥15×10⁷cells/kg of body weight, ≥20×10⁷ cells/kg of body weight, ≥25×10⁷cells/kg of body weight, ≥30×10⁷ cells/kg of body weight, ≥35×10⁷cells/kg of body weight, ≥40×10⁷ cells/kg of body weight, ≥45×10⁷cells/kg of body weight, ≥50×10⁷ cells/kg of body weight, ≥55×10⁷cells/kg of body weight, ≥60×10⁷ cells/kg of body weight, ≥65×10⁷cells/kg of body weight, ≥70×10⁷ cells/kg of body weight, ≥75×10⁷cells/kg of body weight, ≥80×10⁷ cells/kg of body weight, ≥85×10⁷cells/kg of body weight, 90×10⁷ cells/kg of body weight, ≥95×10⁷cells/kg of body weight, ≥10×10⁸ cells/kg of body weight, ≥15×10⁸cells/kg of body weight, ≥20×10⁸ cells/kg of body weight, ≥25×10⁸cells/kg of body weight, ≥30×10⁸ cells/kg of body weight, ≥35×10⁸cells/kg of body weight, ≥40×10⁸ cells/kg of body weight, ≥45×10⁸cells/kg of body weight, ≥50×10⁸ cells/kg of body weight, ≥55×10⁸cells/kg of body weight, ≥60×10⁸ cells/kg of body weight, ≥65×10⁸cells/kg of body weight, ≥70×10⁸ cells/kg of body weight, ≥75×10⁸cells/kg of body weight, ≥80×10⁸ cells/kg of body weight, ≥85×10⁸cells/kg of body weight, 90×10⁸ cells/kg of body weight, ≥95×10⁸cells/kg of body weight or ≥10×10⁹ cells/kg of body weight.

The engineered hematopoietic cells may contain a specific number of CD3+cells for injection into a recipient. In some cases, the target dose ofCD3+ cells to be injected into a recipient is ≥10×10³ cells/kg of bodyweight, ≥15×10³ cells/kg of body weight, ≥20×10³ cells/kg of bodyweight, ≥25×10³ cells/kg of body weight, ≥30×10³ cells/kg of bodyweight, ≥35×10³ cells/kg of body weight, ≥40×10³ cells/kg of bodyweight, ≥45×10³ cells/kg of body weight, ≥50×10³ cells/kg of bodyweight, ≥55×10³ cells/kg of body weight, ≥60×10³ cells/kg of bodyweight, ≥65×10³ cells/kg of body weight, ≥70×10³ cells/kg of bodyweight, ≥75×10³ cells/kg of body weight, ≥80×10³ cells/kg of bodyweight, ≥85×10³ cells/kg of body weight, 90×10³ cells/kg of body weight,≥95×10³ cells/kg of body weight, ≥10×10⁴ cells/kg of body weight,≥15×10⁴ cells/kg of body weight, ≥20×10⁴ cells/kg of body weight,≥25×10⁴ cells/kg of body weight, ≥30×10⁴ cells/kg of body weight,≥35×10⁴ cells/kg of body weight, ≥40×10⁴ cells/kg of body weight,≥45×10⁴ cells/kg of body weight, ≥50×10⁴ cells/kg of body weight,≥55×10⁴ cells/kg of body weight, ≥60×10⁴ cells/kg of body weight,≥65×10⁴ cells/kg of body weight, ≥70×10⁴ cells/kg of body weight,≥75×10⁴ cells/kg of body weight, ≥80×10⁴ cells/kg of body weight,≥85×10⁴ cells/kg of body weight, 90×10⁴ cells/kg of body weight, ≥95×10⁴cells/kg of body weight, ≥10×10⁵ cells/kg of body weight, ≥15×10⁵cells/kg of body weight, ≥20×10⁵ cells/kg of body weight, ≥25×10⁵cells/kg of body weight, ≥30×10⁵ cells/kg of body weight, ≥35×10⁵cells/kg of body weight, ≥40×10⁵ cells/kg of body weight, ≥45×10⁵cells/kg of body weight, ≥50×10⁵ cells/kg of body weight, ≥55×10⁵cells/kg of body weight, ≥60×10⁵ cells/kg of body weight, ≥65×10⁵cells/kg of body weight, ≥70×10⁵ cells/kg of body weight, ≥75×10⁵cells/kg of body weight, ≥80×10⁵ cells/kg of body weight, ≥85×10⁵cells/kg of body weight, 90×10⁵ cells/kg of body weight, ≥95×10⁵cells/kg of body weight, ≥10×10⁶ cells/kg of body weight, ≥15×10⁶cells/kg of body weight, ≥20×10⁶ cells/kg of body weight, ≥25×10⁶cells/kg of body weight, ≥30×10⁶ cells/kg of body weight, ≥35×10⁶cells/kg of body weight, ≥40×10⁶ cells/kg of body weight, ≥45×10⁶cells/kg of body weight, ≥50×10⁶ cells/kg of body weight, ≥55×10⁶cells/kg of body weight, ≥60×10⁶ cells/kg of body weight, ≥65×10⁶cells/kg of body weight, ≥70×10⁶ cells/kg of body weight, ≥75×10⁶cells/kg of body weight, ≥80×10⁶ cells/kg of body weight, ≥85×10⁶cells/kg of body weight, 90×10⁶ cells/kg of body weight, ≥95×10⁶cells/kg of body weight, ≥10×10⁷ cells/kg of body weight, ≥15×10⁷cells/kg of body weight, ≥20×10⁷ cells/kg of body weight, ≥25×10⁷cells/kg of body weight, ≥30×10⁷ cells/kg of body weight, ≥35×10⁷cells/kg of body weight, ≥40×10⁷ cells/kg of body weight, ≥45×10⁷cells/kg of body weight, ≥50×10⁷ cells/kg of body weight, ≥55×10⁷cells/kg of body weight, ≥60×10⁷ cells/kg of body weight, ≥65×10⁷cells/kg of body weight, ≥70×10⁷ cells/kg of body weight, ≥75×10⁷cells/kg of body weight, ≥80×10⁷ cells/kg of body weight, ≥85×10⁷cells/kg of body weight, 90×10⁷ cells/kg of body weight, ≥95×10⁷cells/kg of body weight, ≥10×10⁸ cells/kg of body weight, ≥15×10⁸cells/kg of body weight, ≥20×10⁸ cells/kg of body weight, ≥25×10⁸cells/kg of body weight, ≥30×10⁸ cells/kg of body weight, ≥35×10⁸cells/kg of body weight, ≥40×10⁸ cells/kg of body weight, ≥45×10⁸cells/kg of body weight, ≥50×10⁸ cells/kg of body weight, ≥55×10⁸cells/kg of body weight, ≥60×10⁸ cells/kg of body weight, ≥65×10⁸cells/kg of body weight, ≥70×10⁸ cells/kg of body weight, ≥75×10⁸cells/kg of body weight, ≥80×10⁸ cells/kg of body weight, ≥85×10⁸cells/kg of body weight, 90×10⁸ cells/kg of body weight, ≥95×10⁸cells/kg of body weight or ≥10×10⁹ cells/kg of body weight.

In some cases, the engineered hematopoietic cells may contain acombination of CD34⁺ and CD3⁺ cells for infusion into HLA-matched orHLA-mismatched recipients. In one case, at least 10×10⁶ CD34⁺ cells/kgrecipient weight and at least 1.0×10⁶ CD3⁺ cells/kg are infused into therecipient. In another case, at least 10×10⁶ CD34⁺ cells/kg recipientweight and at least 1.0×10⁷ CD3⁺ cells/kg are infused into therecipient. In another case, at least 10×10⁶ CD34⁺ cells/kg recipientweight and between 1.0-5.0×10⁶ CD3⁺ cells/kg are infused into therecipient. In another case, less than 15×10⁶ CD34⁺ cells/kg recipientweight and at least 50×10⁶ CD3⁺ cells/kg are infused into the recipient.

Isolated and purified CD34+ cells and CD3+ cells may be freshly isolatedor frozen (e.g., cryopreserved) prior to use in an engineeredhematopoietic cell composition. In some cases, the CD34+ cells may becombined with the CD3+ cells prior to use as freshly isolated or frozencells for preparing an engineered hematopoietic cell composition.

In some cases, the CD34+ and CD3+ cells are maintained independentlyeither as freshly isolated cells or as cryopreserved cells. For example,CD34+ cells and CD3+ cells freshly maintained may be combined such thatthe target doses of CD34+ and CD3+ cells are achieved in the engineeredcomposition for infusion. In other cases, CD34+ and CD3+ cellscryopreserved independently may be thawed and the target doses of eachcell type determined after thawing. The thawed CD34+ and CD3+ cells maybe combined such that the target doses of CD34+ and CD3+ cells areachieved in the engineered composition for infusion.

Processing Engineered Hematopoietic Cells for PharmaceuticalCompositions

Engineered hematopoietic cells (e.g., CD34+ and CD3+ cells) may befreshly prepared or previously frozen (e.g., cryopreserved) prior togenerating a pharmaceutical composition for administration to arecipient. In some cases, the CD34+ and CD3+ cells may be HLA-matchedbetween the donor and the recipient. In other cases, the CD34+ and CD3+cells may be HLA-mismatched between the donor and the recipient.

Methods of cryopreservation are described elsewhere herein. In somecases, one aliquot of CD34+ cells is thawed. In other cases, more thanone aliquot of CD34+ cells is thawed. For example, at least one aliquot,two aliquots, three aliquots, four aliquots, five aliquots, sixaliquots, seven aliquots, eight aliquots, nine aliquots, 10 aliquots, 11aliquots, 12 aliquots, 13 aliquots, 14 aliquots, 15 aliquots, 16aliquots, 17 aliquots, 18 aliquots, 19 aliquots, 20 aliquots, 21aliquots, 22 aliquots, 23 aliquots, 24 aliquots, 25 aliquots, 26aliquots, 27 aliquots, 28 aliquots, 29 aliquots, 30 aliquots, 31aliquots, 32 aliquots, 33 aliquots, 34 aliquots, 35 aliquots, 36aliquots, 37 aliquots, 38 aliquots, 39 aliquots, 40 aliquots, 41aliquots, 42 aliquots, 43 aliquots, 44 aliquots, 45 aliquots, 46aliquots, 47 aliquots, 48 aliquots, 49 aliquots, 50 aliquots or morethan 50 aliquots are thawed.

In some cases, one aliquot of CD3+ cells is thawed. In other cases, morethan one aliquot of CD3+ cells is thawed. For example, at least onealiquot, two aliquots, three aliquots, four aliquots, five aliquots, sixaliquots, seven aliquots, eight aliquots, nine aliquots, 10 aliquots, 11aliquots, 12 aliquots, 13 aliquots, 14 aliquots, 15 aliquots, 16aliquots, 17 aliquots, 18 aliquots, 19 aliquots, 20 aliquots, 21aliquots, 22 aliquots, 23 aliquots, 24 aliquots, 25 aliquots, 26aliquots, 27 aliquots, 28 aliquots, 29 aliquots, 30 aliquots, 31aliquots, 32 aliquots, 33 aliquots, 34 aliquots, 35 aliquots, 36aliquots, 37 aliquots, 38 aliquots, 39 aliquots, 40 aliquots, 41aliquots, 42 aliquots, 43 aliquots, 44 aliquots, 45 aliquots, 46aliquots, 47 aliquots, 48 aliquots, 49 aliquots, 50 aliquots or morethan 50 aliquots are thawed.

In some cases, one aliquot of the combination of CD34+ cells and CD3+cells is thawed. In other cases, more than one aliquot of thecombination of CD34+ cells and CD3+ cells is thawed. For example, atleast one aliquot, two aliquots, three aliquots, four aliquots, fivealiquots, six aliquots, seven aliquots, eight aliquots, nine aliquots,10 aliquots, 11 aliquots, 12 aliquots, 13 aliquots, 14 aliquots, 15aliquots, 16 aliquots, 17 aliquots, 18 aliquots, 19 aliquots, 20aliquots, 21 aliquots, 22 aliquots, 23 aliquots, 24 aliquots, 25aliquots, 26 aliquots, 27 aliquots, 28 aliquots, 29 aliquots, 30aliquots, 31 aliquots, 32 aliquots, 33 aliquots, 34 aliquots, 35aliquots, 36 aliquots, 37 aliquots, 38 aliquots, 39 aliquots, 40aliquots, 41 aliquots, 42 aliquots, 43 aliquots, 44 aliquots, 45aliquots, 46 aliquots, 47 aliquots, 48 aliquots, 49 aliquots, 50aliquots or more than 50 aliquots are thawed.

In some cases, freshly prepared engineered hematopoietic cells may beexpanded ex vivo using methods known to those of skill in the art. Inother cases, previously frozen engineered hematopoietic cells may beexpanded ex vivo using methods known to those of skill in the art. Insome cases, either freshly prepared or previously frozen engineeredhematopoietic cells may be expanded ex vivo by use of at least onegrowth factor. In some cases, more than one growth factor may be used toexpand the cells. For example, a growth factor may be activin A,ADAM-10, Angiogenin, Angiopoietin-1, Angiopoietin-2, Angiopoietin-3,Angiopoietin-4, BIO, Bone Morpohogenetic Protien-2, Bone MorpohogeneticProtien-3, Bone Morpohogenetic Protien-4, Bone Morpohogenetic Protien-5,Bone Morpohogenetic Protien-6, Bone Morpohogenetic Protien-7,Brain-derived neurotrophic factor, E-cadherin, Fc chimera, cathepsin G,ch2 inhibitor II, epidermal growth factor, eotaxin, eotaxin-2,eotaxin-3, Fas, fibroblast growth factor-4, fibroblast growth factor-5,fibroblast growth factor-6, fibroblast growth factor-8b, fibroblastgrowth factor-8c, fibroblast growth factor-9, fibroblast growthfactor-10, fibroblast growth factor-17, fibroblast growth factor-18,fibroblast growth factor, fibroblast growth factor acidic, fibroblastgrowth factor basic, fibroblast growth factor basic fragment 1-24bovine, fibroblast growth factor receptor 1a, fibroblast growth factorreceptor 1b, fibroblast growth factor receptor 2a, fibroblast growthfactor receptor 2b fibroblast growth factor receptor 3a, fibroblastgrowth factor receptor 4, flt-3, flk-2 ligand, granulocyte colonystimulating factor, granulocyte-macrophage colony stimulating factor,GROa, GROb, heparin-binding EGF-like growth factor, heregulin-al EGFdomain, heregulin-b1 EGF domain, heregulin B, insulin-like growthfactor-1, insulin-like growth factor-II fragment 33-40, insulin-likegrowth factor binding protein-2, insulin-like growth factor-1,insulin-like growth factor II, interferon a, interferon aA, interferonaA/D, interferon b, interferon g, interferon, interferon g receptor 1,interleukin-1a, interleukin-1b, interleukin soluble receptor type II,interleukin-2, interleukin-2 soluble receptor a, interleukin-2 solublereceptor b, interleukin-2 soluble receptor g, interleukin-3,interleukin-5, interleukin-6, interleukin-6 soluble receptor,interleukin-7, interleukin-8, interleukin-11, interleukin-12, leukemiainhibitory factor, LONG EGF, LONG R2 IGF-1, LYN A, macrophageinflammatory protein-1a, macrophage inflammatory protein-1b, macrophageinflammatory protein-1g, matrix metalloproteinase-1, matrixmetalloproteinase-2, matrix metalloproteinase-9, MIG, monocytechemotactic protein-1, monocyte chemotactic protein-3, monocytechemotactic protein-4, monocyte chemotactic protein-5, nerve growthfactor receptor, neurotrophin-3, neurotrophin-4, noggin, notch-1,oncostatin M, oncostatin M receptor b, osteopontin, osteoprotegrin,phenylarsine oxide, platelet-derived growth factor, platelet-derivedgrowth factor-AB, platelet-derived growth factor-BB, platelet-derivedgrowth factor soluble receptor a, platelet derived growth factorreceptor b, anti-POU5F1, oct4, RANTES, SCF soluble receptor, L-selectin,stem cell factor, stromal cell-derived factor 1a, stromal cell-derivedfactor 1b, thromopoietin, Tie-1, tissue inhibitor ofmetalloproteinase-2, transforming growth factor-a, transforming growthfactor-b1, transforming growth factor-b2, transforming growth factor-b3,transforming growth factor-b1 receptor II soluble fragment, transforminggrowth factor-b soluble receptor III, TrkB, vascular endothelial growthfactor 120, vascular endothelial growth factor 121, vascular endothelialgrowth factor 164, VEGF receptor-2/Flk1/KDR and/or VEGFReceptor-3/Flt-4. The amount of each growth factor used for ex vivoexpansion is known to one of skill in the art and suitable for use withthe methods described herein.

In some cases, either freshly prepared or previously frozen engineeredhematopoietic cells may be expanded ex vivo by use of at least one typeof feeder cell. Any type of feeder cell may be used such that the feedercells maintain viability of engineered hematopoietic cells, and promoteengineered hematopoietic cell proliferation and differentiation. In somecases, at least one growth factor combined with at least one feeder cellmay be used such that the feeder cells maintain viability of engineeredhematopoietic cells, and promote engineered hematopoietic cellproliferation and differentiation. In some cases, feeder cells may bemitotically inactive. In some cases, more than one type of feeder cellmay be used to expand the cells. In some cases, a type of feeder cellmay be derived from adult mouse endothelial cells, embryonic mouseendothelial cells, adult mouse fibroblasts, embryonic mouse fibroblasts,adult human endothelial cells, embryonic human endothelial cells, adulthuman fibroblasts, embryonic human fibroblasts, adult non-human primateendothelial cells, embryonic non-human primate endothelial cells, adultnon-human primate fibroblasts, embryonic non-human primate fibroblasts,adult bovine endothelial cells, embryonic bovine endothelial cells,adult bovine fibroblasts, embryonic bovine fibroblasts, adult porcineendothelial cells, embryonic porcine endothelial cells, adult porcinefibroblasts, embryonic porcine fibroblasts and the like.

In some cases, feeder cells may be modified. For example, themodifications may be genetic. In some cases, feeder cells may expressnon-native genes, repress expression of native genes or overexpressnative genes. For example, feeder cells may express LacZ, GFP, RFP orthe like.

Compositions of Hematopoietic Stem Cells

The hematopoietic stem cells and compositions thereof of the methodsprovided herein can be supplied in the form of a pharmaceuticalcomposition, comprising an isotonic excipient prepared undersufficiently sterile conditions for human administration. Choice of thecellular excipient and any accompanying elements of the composition isadapted in accordance with the route and device used for administration.For general principles in medicinal formulation, the reader is referredto Cell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister& P. Law, Churchill Livingstone, 2000.

In some cases, the hematopoietic stem cells may be HLA-matched betweenthe donor and the recipient. In other cases, the hematopoietic stemcells may be HLA-mismatched between the donor and the recipient.

In some cases, the pharmaceutical composition may contain agents whichenhance engraftment of the hematopoietic cells in the recipient. Inother cases, the pharmaceutical composition may contain agents which donot affect engraftment of the hematopoietic cells in the recipient. Insome cases, the pharmaceutical composition may contain agents whichprevent a negative reaction of the recipient to the hematopoietic cells.For example, any agent as mentioned above may be a cytokine, achemokine, a growth factor, an excipient, a carrier, an inert molecule,an antibody or a fragment thereof, a small molecule, a drug, an agonist,an antagonist, a chemical or the like. Any agent used in apharmaceutical composition of hematopoietic cells in the recipient isphysiologically acceptable.

A variety of methods may be used to deliver hematopoietic cells to therecipient and any method known to one of skill in the art may be appliedto the hematopoietic cells described herein. For example, thehematopoietic cells may be delivered to the recipient by injection usinga needle, catheter, central line or the like. In some cases, thehematopoietic cells may be delivered intravascularly, intravenously,intraarterially, intracranially, intraperitoneally, subcutaneously,intramuscularly, intraorbitally, or through any source which permits thehematopoietic cells to home to an appropriate site in the recipient suchthat the hematopoietic cells persist, regenerate and differentiate inthe recipient.

The composition of engineered hematopoietic cells may also comprise orbe accompanied with one or more other ingredients that facilitate theengraftment or functional mobilization of the cells. For example,ingredients may include matrix proteins that support the cells, promoteadhesion of the cells, or complementary cell types (e.g., endothelialcells).

In some cases, the hematopoietic cells may home to an organ, a tissue ora cell type within the recipient. For example, an organ may the brain,thyroid, eyes, skin, lungs, pancreas, spleen, bladder, prostate,kidneys, stomach, liver, heart, adrenal glands, bronchi, largeintestine, small intestine, spinal cord, bone, bone marrow, pituitarygland, salivary gland, gall bladder, larynx, lymph nodes, prostate,skeletal muscles, appendix, esophagus, parathyroid glands, trachea,urethra, ovaries, testicles, uterus, ureters, fallopian tubes, or anygland in the body. In some cases, a tissue or a cell type may be part ofan organ. In some cases, a tissue or a cell type may be a derived froman organ. In some cases, a tissue or a cell type may be isolated from anorgan.

In some cases, the recipient of the hematopoietic stem cells may nothave received a solid organ transplant. In other cases, the recipientmay have received a solid organ transplant. In some cases, the solidorgan transplant may be performed at least 0.1 days, 0.2 days, 0.3 days,0.4 days, 0.5 days, 0.6 days, 0.7 days, 0.8 days, 0.9 days, 1.0 days,1.1 days, 1.2 days, 1.3 days, 1.4 days, 1.5 days, 1.6 days, 1.7 days,1.8 days, 1.9 days, 2.0 days, 2.1 days, 2.2 days, 2.3 days, 2.4 days,2.5 days, 2.6 days, 2.7 days, 2.8 days, 2.9 days, 3.0 days, 3.1 days,3.2 days, 3.3 days, 3.4 days, 3.5 days, 3.6 days, 3.7 days, 3.8 days,3.9 days, 4.0 days, 4.1 days, 4.2 days, 4.3 days, 4.4 days, 4.5 days,4.6 days, 4.7 days, 4.8 days, 4.9 days, 5.0 days, 5.1 days, 5.2 days,5.3 days, 5.4 days, 5.5 days, 5.6 days, 5.7 days, 5.8 days, 5.9 days,6.0 days, 6.1 days, 6.2 days, 6.3 days, 6.4 days, 6.5 days, 6.6 days,6.7 days, 6.8 days, 6.9 days, 7.0 days, 7.1 days, 7.2 days, 7.3 days,7.4 days, 7.5 days, 7.6 days, 7.7 days, 7.8 days, 7.9 days, 8.0 days,8.1 days, 8.2 days, 8.3 days, 8.4 days, 8.5 days, 8.6 days, 8.7 days,8.8 days, 8.9 days, 9.0 days, 9.1 days, 9.2 days, 9.3 days, 9.4 days,9.5 days, 9.6 days, 9.7 days, 9.8 days, 9.9 days, 10 days, 10.5 days, 11days, 11.5 days, 12 days, 12.5 days, 13 days, 13.5 days, 14 days, 14.5days, 15 days, 15.5 days, 16 days, 16.5 days, 17 days, 17.5 days, 18days, 18.5 days, 19 days, 20 days, 20.5 days, 21 days, 21.5 days, 22days, 22.5 days, 23 days, 23.5 days, 24 days, 24.5 days, 25 days, 25.5days, 26 days, 26.5 days, 27 days, 27.5 days, 28 days, 28.5 days, 29days, 30 days, 30.5 days, 31 days, 31.5 days, 32 days, 32.5 days, 33days, 33.5 days, 34 days, 34.5 days, 35 days, 35.5 days, 36 days, 36.5days, 37 days, 37.5 days, 38 days, 38.5 days, 39 days, 40 days, 40.5days, 41 days, 41.5 days, 42 days, 42.5 days, 43 days, 43.5 days, 44days, 44.5 days, 45 days, 45.5 days, 46 days, 46.5 days, 47 days, 47.5days, 48 days, 48.5 days, 49 days or at least 50 days prior toadministration of the engineered hematopoietic stem cells to therecipient.

In some cases, the solid organ transplant recipient may be administeredone dose of engineered hematopoietic stem cells. In other cases, thesolid organ transplant recipient may be administered more than one doseof engineered hematopoietic stem cells. In some cases, the time elapsedbetween each dose of engineered hematopoietic stem cells may be thesame. In other cases, the time elapsed between each dose of engineeredhematopoietic stem cells may be different.

For example, the solid organ transplant recipient may be administered afirst dose of engineered hematopoietic stem cells at least 0.1 days, 0.2days, 0.3 days, 0.4 days, 0.5 days, 0.6 days, 0.7 days, 0.8 days, 0.9days, 1.0 days, 1.1 days, 1.2 days, 1.3 days, 1.4 days, 1.5 days, 1.6days, 1.7 days, 1.8 days, 1.9 days, 2.0 days, 2.1 days, 2.2 days, 2.3days, 2.4 days, 2.5 days, 2.6 days, 2.7 days, 2.8 days, 2.9 days, 3.0days, 3.1 days, 3.2 days, 3.3 days, 3.4 days, 3.5 days, 3.6 days, 3.7days, 3.8 days, 3.9 days, 4.0 days, 4.1 days, 4.2 days, 4.3 days, 4.4days, 4.5 days, 4.6 days, 4.7 days, 4.8 days, 4.9 days, 5.0 days, 5.1days, 5.2 days, 5.3 days, 5.4 days, 5.5 days, 5.6 days, 5.7 days, 5.8days, 5.9 days, 6.0 days, 6.1 days, 6.2 days, 6.3 days, 6.4 days, 6.5days, 6.6 days, 6.7 days, 6.8 days, 6.9 days, 7.0 days, 7.1 days, 7.2days, 7.3 days, 7.4 days, 7.5 days, 7.6 days, 7.7 days, 7.8 days, 7.9days, 8.0 days, 8.1 days, 8.2 days, 8.3 days, 8.4 days, 8.5 days, 8.6days, 8.7 days, 8.8 days, 8.9 days, 9.0 days, 9.1 days, 9.2 days, 9.3days, 9.4 days, 9.5 days, 9.6 days, 9.7 days, 9.8 days, 9.9 days, 10days, 10.5 days, 11 days, 11.5 days, 12 days, 12.5 days, 13 days, 13.5days, 14 days, 14.5 days, 15 days, 15.5 days, 16 days, 16.5 days, 17days, 17.5 days, 18 days, 18.5 days, 19 days, 20 days, 20.5 days, 21days, 21.5 days, 22 days, 22.5 days, 23 days, 23.5 days, 24 days, 24.5days, 25 days, 25.5 days, 26 days, 26.5 days, 27 days, 27.5 days, 28days, 28.5 days, 29 days, 30 days, 30.5 days, 31 days, 31.5 days, 32days, 32.5 days, 33 days, 33.5 days, 34 days, 34.5 days, 35 days, 35.5days, 36 days, 36.5 days, 37 days, 37.5 days, 38 days, 38.5 days, 39days, 40 days, 40.5 days, 41 days, 41.5 days, 42 days, 42.5 days, 43days, 43.5 days, 44 days, 44.5 days, 45 days, 45.5 days, 46 days, 46.5days, 47 days, 47.5 days, 48 days, 48.5 days, 49 days or at least 50days following solid organ transplantation.

In some cases, a second dose of engineered hematopoietic stem cells maybe administered to the recipient at least In some cases, the solid organtransplant may be performed at least 0.1 days, 0.2 days, 0.3 days, 0.4days, 0.5 days, 0.6 days, 0.7 days, 0.8 days, 0.9 days, 1.0 days, 1.1days, 1.2 days, 1.3 days, 1.4 days, 1.5 days, 1.6 days, 1.7 days, 1.8days, 1.9 days, 2.0 days, 2.1 days, 2.2 days, 2.3 days, 2.4 days, 2.5days, 2.6 days, 2.7 days, 2.8 days, 2.9 days, 3.0 days, 3.1 days, 3.2days, 3.3 days, 3.4 days, 3.5 days, 3.6 days, 3.7 days, 3.8 days, 3.9days, 4.0 days, 4.1 days, 4.2 days, 4.3 days, 4.4 days, 4.5 days, 4.6days, 4.7 days, 4.8 days, 4.9 days, 5.0 days, 5.1 days, 5.2 days, 5.3days, 5.4 days, 5.5 days, 5.6 days, 5.7 days, 5.8 days, 5.9 days, 6.0days, 6.1 days, 6.2 days, 6.3 days, 6.4 days, 6.5 days, 6.6 days, 6.7days, 6.8 days, 6.9 days, 7.0 days, 7.1 days, 7.2 days, 7.3 days, 7.4days, 7.5 days, 7.6 days, 7.7 days, 7.8 days, 7.9 days, 8.0 days, 8.1days, 8.2 days, 8.3 days, 8.4 days, 8.5 days, 8.6 days, 8.7 days, 8.8days, 8.9 days, 9.0 days, 9.1 days, 9.2 days, 9.3 days, 9.4 days, 9.5days, 9.6 days, 9.7 days, 9.8 days, 9.9 days, 10 days, 10.5 days, 11days, 11.5 days, 12 days, 12.5 days, 13 days, 13.5 days, 14 days, 14.5days, 15 days, 15.5 days, 16 days, 16.5 days, 17 days, 17.5 days, 18days, 18.5 days, 19 days, 20 days, 20.5 days, 21 days, 21.5 days, 22days, 22.5 days, 23 days, 23.5 days, 24 days, 24.5 days, 25 days, 25.5days, 26 days, 26.5 days, 27 days, 27.5 days, 28 days, 28.5 days, 29days, 30 days, 30.5 days, 31 days, 31.5 days, 32 days, 32.5 days, 33days, 33.5 days, 34 days, 34.5 days, 35 days, 35.5 days, 36 days, 36.5days, 37 days, 37.5 days, 38 days, 38.5 days, 39 days, 40 days, 40.5days, 41 days, 41.5 days, 42 days, 42.5 days, 43 days, 43.5 days, 44days, 44.5 days, 45 days, 45.5 days, 46 days, 46.5 days, 47 days, 47.5days, 48 days, 48.5 days, 49 days or at least 50 days after the firstdose of engineered hematopoietic stem cells such that administration ofengineered hematopoietic stem cells is recursive.

In some cases, more than two doses of engineered hematopoietic stemcells may be administered to the solid organ transplant recipient. Forexample, at least 0.1 days, 0.2 days, 0.3 days, 0.4 days, 0.5 days, 0.6days, 0.7 days, 0.8 days, 0.9 days, 1.0 days, 1.1 days, 1.2 days, 1.3days, 1.4 days, 1.5 days, 1.6 days, 1.7 days, 1.8 days, 1.9 days, 2.0days, 2.1 days, 2.2 days, 2.3 days, 2.4 days, 2.5 days, 2.6 days, 2.7days, 2.8 days, 2.9 days, 3.0 days, 3.1 days, 3.2 days, 3.3 days, 3.4days, 3.5 days, 3.6 days, 3.7 days, 3.8 days, 3.9 days, 4.0 days, 4.1days, 4.2 days, 4.3 days, 4.4 days, 4.5 days, 4.6 days, 4.7 days, 4.8days, 4.9 days, 5.0 days, 5.1 days, 5.2 days, 5.3 days, 5.4 days, 5.5days, 5.6 days, 5.7 days, 5.8 days, 5.9 days, 6.0 days, 6.1 days, 6.2days, 6.3 days, 6.4 days, 6.5 days, 6.6 days, 6.7 days, 6.8 days, 6.9days, 7.0 days, 7.1 days, 7.2 days, 7.3 days, 7.4 days, 7.5 days, 7.6days, 7.7 days, 7.8 days, 7.9 days, 8.0 days, 8.1 days, 8.2 days, 8.3days, 8.4 days, 8.5 days, 8.6 days, 8.7 days, 8.8 days, 8.9 days, 9.0days, 9.1 days, 9.2 days, 9.3 days, 9.4 days, 9.5 days, 9.6 days, 9.7days, 9.8 days, 9.9 days, 10 days, 10.5 days, 11 days, 11.5 days, 12days, 12.5 days, 13 days, 13.5 days, 14 days, 14.5 days, 15 days, 15.5days, 16 days, 16.5 days, 17 days, 17.5 days, 18 days, 18.5 days, 19days, 20 days, 20.5 days, 21 days, 21.5 days, 22 days, 22.5 days, 23days, 23.5 days, 24 days, 24.5 days, 25 days, 25.5 days, 26 days, 26.5days, 27 days, 27.5 days, 28 days, 28.5 days, 29 days, 30 days, 30.5days, 31 days, 31.5 days, 32 days, 32.5 days, 33 days, 33.5 days, 34days, 34.5 days, 35 days, 35.5 days, 36 days, 36.5 days, 37 days, 37.5days, 38 days, 38.5 days, 39 days, 40 days, 40.5 days, 41 days, 41.5days, 42 days, 42.5 days, 43 days, 43.5 days, 44 days, 44.5 days, 45days, 45.5 days, 46 days, 46.5 days, 47 days, 47.5 days, 48 days, 48.5days, 49 days or at least 50 days may pass between administration of thesecond and the third doses of engineered hematopoietic stem cells suchthat administration of engineered hematopoietic stem cells is recursive.

Any of the above mentioned time frames may also pass between the thirdand fourth doses, the fourth and fifth doses, the fifth and sixth doses,the sixth and seventh doses, the seventh and eighth doses, the eighthand ninth doses, the ninth and tenth doses and so on.

Non-Myeloablative Conditioning

Following transplantation of the HLA-matched or HLA-mismatched solidorgan, the recipient may be treated with non-myeloablative conditioning.In some cases, non-myeloablative conditioning may be performed usingmethods known to those of skill in the art. In other cases, recipientsmay receive non-myeloablative conditioning that includes a plurality ofagents. In some cases, the agents may include thymoglobulin (ATG), a Tcell depleting agent and/or radiation.

In some cases, ATG may be delivered intravenously. In some cases, asingle dose of ATG may be delivered to the recipient. In other cases,the recipient may receive more than one dose of ATG. For example, arecipient may receive at least one dose of ATG, two doses of ATG, threedoses of ATG, four doses of ATG, five doses of ATG, six doses of ATG,seven doses of ATG, eight doses of ATG, nine doses of ATG, 10 doses ofATG, 11 doses of ATG, 12 doses of ATG, 13 doses of ATG, 14 doses of ATG,15 doses of ATG, 16 doses of ATG, 17 doses of ATG, 18 doses of ATG, 19doses of ATG, or at least 20 doses of ATG.

In some cases, each dose of ATG may be at least 0.1 mg/kg, 0.2 mg/kg,0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg,1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg,2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1 mg/kg,4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg, 4.8mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg, 5.4 mg/kg,5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg, 6.0 mg/kg, 6.1mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7 mg/kg,6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg, 7.4mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg, 8.0 mg/kg,8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6 mg/kg, 8.7mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3 mg/kg,9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg, 10mg/kg, 10.5 mg/kg, 11 mg/kg, 11.5 mg/kg, 12 mg/kg, 12.5 mg/kg, 13 mg/kg,13.5 mg/kg, 14 mg/kg, 14.5 mg/kg, 15 mg/kg, 15.5 mg/kg, 16 mg/kg, 16.5mg/kg, 17 mg/kg, 17.5 mg/kg, 18 mg/kg, 18.5 mg/kg, 19 mg/kg or at least20 mg/kg.

ATG may be administered on the same day of solid-organ transplantation.In some cases, the plurality of ATG doses may be administered over aperiod of time after organ transplantation. In some cases, the pluralityof ATG doses may be administered over a period of at least 0.1 days, 0.2days, 0.3 days, 0.4 days, 0.5 days, 0.6 days, 0.7 days, 0.8 days, 0.9days, 1.0 days, 1.1 days, 1.2 days, 1.3 days, 1.4 days, 1.5 days, 1.6days, 1.7 days, 1.8 days, 1.9 days, 2.0 days, 2.1 days, 2.2 days, 2.3days, 2.4 days, 2.5 days, 2.6 days, 2.7 days, 2.8 days, 2.9 days, 3.0days, 3.1 days, 3.2 days, 3.3 days, 3.4 days, 3.5 days, 3.6 days, 3.7days, 3.8 days, 3.9 days, 4.0 days, 4.1 days, 4.2 days, 4.3 days, 4.4days, 4.5 days, 4.6 days, 4.7 days, 4.8 days, 4.9 days, 5.0 days, 5.1days, 5.2 days, 5.3 days, 5.4 days, 5.5 days, 5.6 days, 5.7 days, 5.8days, 5.9 days, 6.0 days, 6.1 days, 6.2 days, 6.3 days, 6.4 days, 6.5days, 6.6 days, 6.7 days, 6.8 days, 6.9 days, 7.0 days, 7.1 days, 7.2days, 7.3 days, 7.4 days, 7.5 days, 7.6 days, 7.7 days, 7.8 days, 7.9days, 8.0 days, 8.1 days, 8.2 days, 8.3 days, 8.4 days, 8.5 days, 8.6days, 8.7 days, 8.8 days, 8.9 days, 9.0 days, 9.1 days, 9.2 days, 9.3days, 9.4 days, 9.5 days, 9.6 days, 9.7 days, 9.8 days, 9.9 days, 10days, 10.5 days, 11 days, 11.5 days, 12 days, 12.5 days, 13 days, 13.5days, 14 days, 14.5 days, 15 days, 15.5 days, 16 days, 16.5 days, 17days, 17.5 days, 18 days, 18.5 days, 19 days or at least 20 days.

In some cases, the ATG is delivered intra-operatively before thetransplanted organ is perfused with host blood. In other cases, the ATGis delivered intra-operatively after the transplanted organ is perfusedwith host blood. In some cases, the ATG is delivered intra-venouslybefore the transplanted organ is perfused with host blood. In othercases, the ATG is delivered intra-venously after the transplanted organis perfused with host blood. In some cases, the ATG is deliveredintra-arterially before the transplanted organ is perfused with hostblood. In other cases, the ATG is delivered intra-arterially after thetransplanted organ is perfused with host blood. In some cases, the ATGis delivered subcutaneously before the transplanted organ is perfusedwith host blood. In other cases, the ATG is delivered subcutaneouslyafter the transplanted organ is perfused with host blood. In some cases,the ATG is delivered intraperitoneally before the transplanted organ isperfused with host blood. In other cases, the ATG is deliveredintraperitonially after the transplanted organ is perfused with hostblood.

Corticosteroid therapy may be given as medication prior toadministration of ATG. In some cases, solumedrol may be administeredalthough any corticosteroid known to one of skill in the art sufficientto reduce side effects of ATG may be used at an effective dose. In somecases, the corticosterioid may be administered on the same day as ATG isadministered. For example, solumedrol may be administered at a dosewithin the range of 0-40 mg, 5-50 mg, 10-60 mg, 15-65 mg, 20-70 mg,25-75 mg, 30-80 mg, 35-85 mg, 40-90 mg, 45-95 mg, 50-100 mg, 55-105 mg,60-110 mg, 65-115 mg, 70-120 mg, 75-125 mg, 80-130 mg, 85-135 mg, 90-140mg, 95-145 mg, 100-150 mg, 105-155 mg, 110-160 mg, 115-165 mg, 120-170mg, 125-175 mg, 130-180 mg, 135-185 mg, 140-190 mg, 145-195 mg or150-200 mg.

Following the final dose of ATG administered to the recipient,prednisone may be administered. In some cases, a single dose ofprednisone may be administered. In other cases, more than one dose ofprednisone may be administered. For example, multiple doses ofprednisone may be administered according to a tapering course or aconstant course.

In some cases, for a tapering course, the first dose of prednisone maystart at 100 mg/d and then the dose reduced by 5 mg/d until constant at5 mg/d for at least 15 days, the first dose of prednisone may start at90 mg/d and reduced by 5 mg/d until constant for at least 15 days, thefirst dose of prednisone may start at 80 mg/d and reduced by 5 mg/duntil constant for at least 15 days, the first dose of prednisone maystart at 70 mg/d and reduced by 5 mg/d until constant for at least 15days, the first dose of prednisone may start at 60 mg/d and reduced by 5mg/d until constant for at least 15 days, the first dose of prednisonemay start at 50 mg/d and reduced by 5 mg/d until constant for at least15 days, the first dose of prednisone may start at 40 mg/d and reducedby 5 mg/d until constant for at least 15 days, the first dose ofprednisone may start at 30 mg/d and reduced by 5 mg/d until constant forat least 15 days, the first dose of prednisone may start at 20 mg/d andreduced by 5 mg/d until constant for at least 15 days or the first doseof prednisone may start at 10 mg/d and reduced by 5 mg/d until constantfor at least 15 days. In some cases, for a constant course, the doses ofprednisone may be 100 mg/d, 90 mg/d, 80 mg/d, 70 mg/d, 60 mg/d, 50 mg/d,40 mg/d, 30 mg/d, 20 mg/d, 10 mg/d or 5 mg/d for at least 15 days.

In some cases, for a tapering course, the first dose of prednisone maystart at 100 mg/d and reduced by 5 mg/d until constant for at least 30days, the first dose of prednisone may start at 90 mg/d and reduced by 5mg/d until constant for at least 30 days, the first dose of prednisonemay start at 80 mg/d and reduced by 5 mg/d until constant for at least30 days, the first dose of prednisone may start at 70 mg/d and reducedby 5 mg/d until constant for at least 30 days, the first dose ofprednisone may start at 60 mg/d and reduced by 5 mg/d until constant forat least 30 days, the first dose of prednisone may start at 50 mg/d andreduced by 5 mg/d until constant for at least 30 days, the first dose ofprednisone may start at 40 mg/d and reduced by 5 mg/d until constant forat least 30 days, the first dose of prednisone may start at 30 mg/d andreduced by 5 mg/d until constant for at least 30 days, the first dose ofprednisone may start at 20 mg/d and reduced by 5 mg/d until constant forat least 30 days or the first dose of prednisone may start at 10 mg/dand reduced by 5 mg/d until constant for at least 30 days. In somecases, for a constant course, the doses of prednisone may be 100 mg/d,90 mg/d, 80 mg/d, 70 mg/d, 60 mg/d, 50 mg/d, 40 mg/d, 30 mg/d, 20 mg/d,10 mg/d or 5 mg/d for at least 30 days.

In some cases, for a tapering course, the first dose of prednisone maystart at 100 mg/d and reduced by 5 mg/d until constant for at least 45days, the first dose of prednisone may start at 90 mg/d and reduced by 5mg/d until constant for at least 45 days, the first dose of prednisonemay start at 80 mg/d and reduced by 5 mg/d until constant for at least45 days, the first dose of prednisone may start at 70 mg/d and reducedby 5 mg/d until constant for at least 45 days, the first dose ofprednisone may start at 60 mg/d and reduced by 5 mg/d until constant forat least 45 days, the first dose of prednisone may start at 50 mg/d andreduced by 5 mg/d until constant for at least 45 days, the first dose ofprednisone may start at 40 mg/d and reduced by 5 mg/d until constant forat least 45 days, the first dose of prednisone may start at 30 mg/d andreduced by 5 mg/d until constant for at least 45 days, the first dose ofprednisone may start at 20 mg/d and reduced by 5 mg/d until constant forat least 45 days or the first dose of prednisone may start at 10 mg/dand reduced by 5 mg/d until constant for at least 45 days. In somecases, for a constant course, the doses of prednisone may be 100 mg/d,90 mg/d, 80 mg/d, 70 mg/d, 60 mg/d, 50 mg/d, 40 mg/d, 30 mg/d, 20 mg/d,10 mg/d or 5 mg/d for at least 45 days.

In some cases, for a tapering course, the first dose of prednisone maystart at 100 mg/d and reduced by 5 mg/d until constant for at least 60days, the first dose of prednisone may start at 90 mg/d and reduced by 5mg/d until constant for at least 60 days, the first dose of prednisonemay start at 80 mg/d and reduced by 5 mg/d until constant for at least60 days, the first dose of prednisone may start at 70 mg/d and reducedby 5 mg/d until constant for at least 60 days, the first dose ofprednisone may start at 60 mg/d and reduced by 5 mg/d until constant forat least 60 days, the first dose of prednisone may start at 50 mg/d andreduced by 5 mg/d until constant for at least 60 days, the first dose ofprednisone may start at 40 mg/d and reduced by 5 mg/d until constant forat least 60 days, the first dose of prednisone may start at 30 mg/d andreduced by 5 mg/d until constant for at least 60 days, the first dose ofprednisone may start at 20 mg/d and reduced by 5 mg/d until constant forat least 60 days or the first dose of prednisone may start at 10 mg/dand reduced by 5 mg/d until constant for at least 60 days. In somecases, for a constant course, the doses of prednisone may be 100 mg/d,90 mg/d, 80 mg/d, 70 mg/d, 60 mg/d, 50 mg/d, 40 mg/d, 30 mg/d, 20 mg/d,10 mg/d or 5 mg/d for at least 60 days.

The corticosteroid and/or prednisone may be administeredintravascularly, intravenously, intraarterially, intracranially,intraperitoneally, subcutaneously, intramuscularly, intraorbitally,orally, topically, or through any source which permits proper metabolismof the corticosteroid and/or prednisone by the recipient.

In some cases, any T cell depleting agent known to one of skill in theart can be used as a portion of a non-myeloablative conditioning regimefor the recipient. In some cases, the T cell depleting agent may be ananti-T cell monoclonal antibody or a T cell depleting drug (e.g.,fludarabine). In some cases, a single T cell depleting agent isadministered to the recipient. In other cases, more than one T celldepleting agent is administered to the recipient.

In some cases, a T cell depleting agent may be delivered intravenously.In some cases, a single dose of a T cell depleting agent may bedelivered to the recipient. In other cases, the recipient may receivemore than one dose of a T cell depleting agent. For example, a recipientmay receive at least one dose of a T cell depleting agent, two doses ofa T cell depleting agent, three doses of a T cell depleting agent, fourdoses of a T cell depleting agent, five doses of a T cell depletingagent, six doses of a T cell depleting agent, seven doses of a T celldepleting agent, eight doses of a T cell depleting agent, nine doses ofa T cell depleting agent, 10 doses of a T cell depleting agent, 11 dosesof a T cell depleting agent, 12 doses of a T cell depleting agent, 13doses of a T cell depleting agent, 14 doses of a T cell depleting agent,15 doses of a T cell depleting agent, 16 doses of a T cell depletingagent, 17 doses of a T cell depleting agent, 18 doses of a T celldepleting agent, 19 doses of a T cell depleting agent, or 20 doses of aT cell depleting agent.

A T cell depleting agent may be administered on the same day ofsolid-organ transplantation. In some cases, the plurality of T celldepleting agent doses may be delivered over a period of time after organtransplantation. In some cases, the plurality of T cell depleting agentdoses may be delivered over a period of at least 0.1 days, 0.2 days, 0.3days, 0.4 days, 0.5 days, 0.6 days, 0.7 days, 0.8 days, 0.9 days, 1.0days, 1.1 days, 1.2 days, 1.3 days, 1.4 days, 1.5 days, 1.6 days, 1.7days, 1.8 days, 1.9 days, 2.0 days, 2.1 days, 2.2 days, 2.3 days, 2.4days, 2.5 days, 2.6 days, 2.7 days, 2.8 days, 2.9 days, 3.0 days, 3.1days, 3.2 days, 3.3 days, 3.4 days, 3.5 days, 3.6 days, 3.7 days, 3.8days, 3.9 days, 4.0 days, 4.1 days, 4.2 days, 4.3 days, 4.4 days, 4.5days, 4.6 days, 4.7 days, 4.8 days, 4.9 days, 5.0 days, 5.1 days, 5.2days, 5.3 days, 5.4 days, 5.5 days, 5.6 days, 5.7 days, 5.8 days, 5.9days, 6.0 days, 6.1 days, 6.2 days, 6.3 days, 6.4 days, 6.5 days, 6.6days, 6.7 days, 6.8 days, 6.9 days, 7.0 days, 7.1 days, 7.2 days, 7.3days, 7.4 days, 7.5 days, 7.6 days, 7.7 days, 7.8 days, 7.9 days, 8.0days, 8.1 days, 8.2 days, 8.3 days, 8.4 days, 8.5 days, 8.6 days, 8.7days, 8.8 days, 8.9 days, 9.0 days, 9.1 days, 9.2 days, 9.3 days, 9.4days, 9.5 days, 9.6 days, 9.7 days, 9.8 days, 9.9 days, 10 days, 10.5days, 11 days, 11.5 days, 12 days, 12.5 days, 13 days, 13.5 days, 14days, 14.5 days, 15 days, 15.5 days, 16 days, 16.5 days, 17 days, 17.5days, 18 days, 18.5 days, 19 days or at least 20 days.

In some cases, the T cell depleting agent is delivered intra-operativelybefore the transplanted organ is perfused with host blood. In othercases, the T cell depleting agent is delivered intra-operatively afterthe transplanted organ is perfused with host blood. In some cases, the Tcell depleting agent is delivered intravenously before the transplantedorgan is perfused with host blood. In other cases, the T cell depletingagent is delivered intravenously after the transplanted organ isperfused with host blood. In some cases, the T cell depleting agent isdelivered intra-arterially before the transplanted organ is perfusedwith host blood. In other cases, the T cell depleting agent is deliveredintra-arterially after the transplanted organ is perfused with hostblood. In some cases, the T cell depleting agent is deliveredsubcutaneously before the transplanted organ is perfused with hostblood. In other cases, the T cell depleting agent is deliveredsubcutaneously after the transplanted organ is perfused with host blood.In some cases, the T cell depleting agent is delivered intraperitoneallybefore the transplanted organ is perfused with host blood. In othercases, the T cell depleting agent is delivered intraperitoneally afterthe transplanted organ is perfused with host blood.

In some cases, fludarabine may be delivered intravenously. In somecases, a single dose of fludarabine may be delivered to the recipient.In other cases, the recipient may receive more than one dose offludarabine. For example, a recipient may receive at least one dose offludarabine, two doses of fludarabine, three doses of fludarabine, fourdoses of fludarabine, five doses of fludarabine, six doses offludarabine, seven doses of fludarabine, eight doses of fludarabine,nine doses of fludarabine, 10 doses of fludarabine, 11 doses offludarabine, 12 doses of fludarabine, 13 doses of fludarabine, 14 dosesof fludarabine, 15 doses of fludarabine, 16 doses of fludarabine, 17doses of fludarabine, 18 doses of fludarabine, 19 doses of fludarabine,or at least 20 doses of fludarabine.

In some cases, each dose of fludarabine may be at least 0.1 mg/m2/d, 0.2mg/m2/d, 0.3 mg/m2/d, 0.4 mg/m2/d, 0.5 mg/m2/d, 0.6 mg/m2/d, 0.7mg/m2/d, 0.8 mg/m2/d, 0.9 mg/m2/d, 1.0 mg/m2/d, 1.1 mg/m2/d, 1.2mg/m2/d, 1.3 mg/m2/d, 1.4 mg/m2/d, 1.5 mg/m2/d, 1.6 mg/m2/d, 1.7mg/m2/d, 1.8 mg/m2/d, 1.9 mg/m2/d, 2.0 mg/m2/d, 2.1 mg/m2/d, 2.2mg/m2/d, 2.3 mg/m2/d, 2.4 mg/m2/d, 2.5 mg/m2/d, 2.6 mg/m2/d, 2.7mg/m2/d, 2.8 mg/m2/d, 2.9 mg/m2/d, 3.0 mg/m2/d, 3.1 mg/m2/d, 3.2mg/m2/d, 3.3 mg/m2/d, 3.4 mg/m2/d, 3.5 mg/m2/d, 3.6 mg/m2/d, 3.7mg/m2/d, 3.8 mg/m2/d, 3.9 mg/m2/d, 4.0 mg/m2/d, 4.1 mg/m2/d, 4.2mg/m2/d, 4.3 mg/m2/d, 4.4 mg/m2/d, 4.5 mg/m2/d, 4.6 mg/m2/d, 4.7mg/m2/d, 4.8 mg/m2/d, 4.9 mg/m2/d, 5.0 mg/m2/d, 5.1 mg/m2/d, 5.2mg/m2/d, 5.3 mg/m2/d, 5.4 mg/m2/d, 5.5 mg/m2/d, 5.6 mg/m2/d, 5.7mg/m2/d, 5.8 mg/m2/d, 5.9 mg/m2/d, 6.0 mg/m2/d, 6.1 mg/m2/d, 6.2mg/m2/d, 6.3 mg/m2/d, 6.4 mg/m2/d, 6.5 mg/m2/d, 6.6 mg/m2/d, 6.7mg/m2/d, 6.8 mg/m2/d, 6.9 mg/m2/d, 7.0 mg/m2/d, 7.1 mg/m2/d, 7.2mg/m2/d, 7.3 mg/m2/d, 7.4 mg/m2/d, 7.5 mg/m2/d, 7.6 mg/m2/d, 7.7mg/m2/d, 7.8 mg/m2/d, 7.9 mg/m2/d, 8.0 mg/m2/d, 8.1 mg/m2/d, 8.2mg/m2/d, 8.3 mg/m2/d, 8.4 mg/m2/d, 8.5 mg/m2/d, 8.6 mg/m2/d, 8.7mg/m2/d, 8.8 mg/m2/d, 8.9 mg/m2/d, 9.0 mg/m2/d, 9.1 mg/m2/d, 9.2mg/m2/d, 9.3 mg/m2/d, 9.4 mg/m2/d, 9.5 mg/m2/d, 9.6 mg/m2/d, 9.7mg/m2/d, 9.8 mg/m2/d, 9.9 mg/m2/d, 10 mg/m2/d, 10.5 mg/m2/d, 11 mg/m2/d,11.5 mg/m2/d, 12 mg/m2/d, 12.5 mg/m2/d, 13 mg/m2/d, 13.5 mg/m2/d, 14mg/m2/d, 14.5 mg/m2/d, 15 mg/m2/d, 15.5 mg/m2/d, 16 mg/m2/d, 16.5mg/m2/d, 17 mg/m2/d, 17.5 mg/m2/d, 18 mg/m2/d, 18.5 mg/m2/d, 19 mg/m2/d,20 mg/m2/d, 20.5 mg/m2/d, 21 mg/m2/d, 21.5 mg/m2/d, 22 mg/m2/d, 22.5mg/m2/d, 23 mg/m2/d, 23.5 mg/m2/d, 24 mg/m2/d, 24.5 mg/m2/d, 25 mg/m2/d,25.5 mg/m2/d, 26 mg/m2/d, 26.5 mg/m2/d, 27 mg/m2/d, 27.5 mg/m2/d, 28mg/m2/d, 28.5 mg/m2/d, 29 mg/m2/d, 30 mg/m2/d, 30.5 mg/m2/d, 31 mg/m2/d,31.5 mg/m2/d, 32 mg/m2/d, 32.5 mg/m2/d, 33 mg/m2/d, 33.5 mg/m2/d, 34mg/m2/d, 34.5 mg/m2/d, 35 mg/m2/d, 35.5 mg/m2/d, 36 mg/m2/d, 36.5mg/m2/d, 37 mg/m2/d, 37.5 mg/m2/d, 38 mg/m2/d, 38.5 mg/m2/d, 39 mg/m2/d,4 0 mg/m2/d, 40.5 mg/m2/d, 41 mg/m2/d, 41.5 mg/m2/d, 42 mg/m2/d, 42.5mg/m2/d, 43 mg/m2/d, 43.5 mg/m2/d, 44 mg/m2/d, 44.5 mg/m2/d, 45 mg/m2/d,45.5 mg/m2/d, 46 mg/m2/d, 46.5 mg/m2/d, 47 mg/m2/d, 47.5 mg/m2/d, 48mg/m2/d, 48.5 mg/m2/d, 49 mg/m2/d, 50 mg/m2/d, 50.5 mg/m2/d, 51 mg/m2/d,51.5 mg/m2/d, 52 mg/m2/d, 52.5 mg/m2/d, 53 mg/m2/d, 53.5 mg/m2/d, 54mg/m2/d, 54.5 mg/m2/d, 55 mg/m2/d, 55.5 mg/m2/d, 56 mg/m2/d, 56.5mg/m2/d, 57 mg/m2/d, 57.5 mg/m2/d, 58 mg/m2/d, 58.5 mg/m2/d, 59 mg/m2/dor at least 60 mg/m2/d.

Fludarabine may be administered on the same day of solid-organtransplantation. In some cases, the plurality of fludarabine doses maybe delivered over a period of time after organ transplantation. In somecases, the plurality of fludarabine doses may be delivered over a periodof at least 0.1 days, 0.2 days, 0.3 days, 0.4 days, 0.5 days, 0.6 days,0.7 days, 0.8 days, 0.9 days, 1.0 days, 1.1 days, 1.2 days, 1.3 days,1.4 days, 1.5 days, 1.6 days, 1.7 days, 1.8 days, 1.9 days, 2.0 days,2.1 days, 2.2 days, 2.3 days, 2.4 days, 2.5 days, 2.6 days, 2.7 days,2.8 days, 2.9 days, 3.0 days, 3.1 days, 3.2 days, 3.3 days, 3.4 days,3.5 days, 3.6 days, 3.7 days, 3.8 days, 3.9 days, 4.0 days, 4.1 days,4.2 days, 4.3 days, 4.4 days, 4.5 days, 4.6 days, 4.7 days, 4.8 days,4.9 days, 5.0 days, 5.1 days, 5.2 days, 5.3 days, 5.4 days, 5.5 days,5.6 days, 5.7 days, 5.8 days, 5.9 days, 6.0 days, 6.1 days, 6.2 days,6.3 days, 6.4 days, 6.5 days, 6.6 days, 6.7 days, 6.8 days, 6.9 days,7.0 days, 7.1 days, 7.2 days, 7.3 days, 7.4 days, 7.5 days, 7.6 days,7.7 days, 7.8 days, 7.9 days, 8.0 days, 8.1 days, 8.2 days, 8.3 days,8.4 days, 8.5 days, 8.6 days, 8.7 days, 8.8 days, 8.9 days, 9.0 days,9.1 days, 9.2 days, 9.3 days, 9.4 days, 9.5 days, 9.6 days, 9.7 days,9.8 days, 9.9 days, 10 days, 10.5 days, 11 days, 11.5 days, 12 days,12.5 days, 13 days, 13.5 days, 14 days, 14.5 days, 15 days, 15.5 days,16 days, 16.5 days, 17 days, 17.5 days, 18 days, 18.5 days, 19 days orat least 20 days.

In some cases, the fludarabine is delivered intra-operatively before thetransplanted organ is perfused with host blood. In other cases, thefludarabine is delivered intra-operatively after the transplanted organis perfused with host blood. In some cases, the fludarabine is deliveredintra-venously before the transplanted organ is perfused with hostblood. In other cases, the fludarabine is delivered intra-venously afterthe transplanted organ is perfused with host blood. In some cases, thefludarabine is delivered intra-arterially before the transplanted organis perfused with host blood. In other cases, the fludarabine isdelivered intra-arterially after the transplanted organ is perfused withhost blood. In some cases, the fludarabine is delivered subcutaneouslybefore the transplanted organ is perfused with host blood. In othercases, the fludarabine is delivered subcutaneously after thetransplanted organ is perfused with host blood. In some cases, thefludarabine is delivered intraperitoneally before the transplanted organis perfused with host blood. In other cases, the fludarabine isdelivered intraperitoneally after the transplanted organ is perfusedwith host blood.

In some cases, recipients are treated with irradiation. The irradiationmay be fractionated or unfractionated. In the case that a recipient istreated with more than one dose of irradiation, all doses may befractionated. In another case that a recipient is treated with more thanone dose of irradiation, all doses may be unfractionated. In anothercase that a recipient is treated with more than one dose of irradiation,the doses may be a mix of fractionated unfractionated.

In some cases, the irradiation is delivered intraoperatively. In somecases, the irradiation is delivered intravenously. In some cases, theirradiation is delivered intraarterially. In some cases, the irradiationis delivered subcutaneously. In some cases, the irradiation is deliveredintraperitoneally.

In some cases, a single dose of irradiation may be delivered to therecipient. In other cases, the recipient may receive more than one doseof irradiation. For example, a recipient may receive at least one doseof irradiation, two doses of irradiation, three doses of irradiation,four doses of irradiation, five doses of irradiation, six doses ofirradiation, seven doses of irradiation, eight doses of irradiation,nine doses of irradiation, 10 doses of irradiation, 11 doses ofirradiation, 12 doses of irradiation, 13 doses of irradiation, 14 dosesof irradiation, 15 doses of irradiation, 16 doses of irradiation, 17doses of irradiation, 18 doses of irradiation, 19 doses of irradiation,or at least 20 doses of irradiation.

In some cases, each dose of irradiation may be at least 1 cGy, 2 cGy, 3cGy, 4 cGy, 5 cGy, 6 cGy, 7 cGy, 8 cGy, 9 cGy, 10 cGy, 11 cGy, 12 cGy,13 cGy, 14 cGy, 15 cGy, 16 cGy, 17 cGy, 18 cGy, 19 cGy, 20 cGy, 21 cGy,22 cGy, 23 cGy, 24 cGy, 25 cGy, 26 cGy, 27 cGy, 28 cGy, 29 cGy, 30 cGy,31 cGy, 32 cGy, 33 cGy, 34 cGy, 35 cGy, 36 cGy, 37 cGy, 38 cGy, 39 cGy,40 cGy, 41 cGy, 42 cGy, 43 cGy, 44 cGy, 45 cGy, 46 cGy, 47 cGy, 48 cGy,49 cGy, 50 cGy, 51 cGy, 52 cGy, 53 cGy, 54 cGy, 55 cGy, 56 cGy, 57 cGy,58 cGy, 59 cGy, 60 cGy, 61 cGy, 62 cGy, 63 cGy, 64 cGy, 65 cGy, 66 cGy,67 cGy, 68 cGy, 69 cGy, 70 cGy, 71 cGy, 72 cGy, 73 cGy, 74 cGy, 75 cGy,76 cGy, 77 cGy, 78 cGy, 79 cGy, 80 cGy, 81 cGy, 82 cGy, 83 cGy, 84 cGy,85 cGy, 86 cGy, 87 cGy, 88 cGy, 89 cGy, 90 cGy, 91 cGy, 92 cGy, 93 cGy,94 cGy, 95 cGy, 96 cGy, 97 cGy, 98 cGy, 99 cGy, 100 cGy, 105 cGy, 110cGy, 115 cGy, 120 cGy, 125 cGy, 130 cGy, 135 cGy, 140 cGy, 145 cGy, 150cGy, 155 cGy, 160 cGy, 165 cGy, 170 cGy, 175 cGy, 180 cGy, 185 cGy, 190cGy, 195 cGy, 200 cGy, 205 cGy, 210 cGy, 215 cGy, 220 cGy, 225 cGy, 230cGy, 235 cGy, 240 cGy, 245 cGy, 250 cGy, 255 cGy, 260 cGy, 265 cGy, 270cGy, 275 cGy, 280 cGy, 285 cGy, 290 cGy, 295 cGy, 300 cGy, 305 cGy, 310cGy, 315 cGy, 320 cGy, 325 cGy, 330 cGy, 335 cGy, 340 cGy, 345 cGy, 350cGy, 355 cGy, 360 cGy, 365 cGy, 370 cGy, 375 cGy, 380 cGy, 385 cGy, 390cGy, 395 cGy, 400 cGy, 405 cGy, 410 cGy, 415 cGy, 420 cGy, 425 cGy, 430cGy, 435 cGy, 440 cGy, 445 cGy, 450 cGy, 455 cGy, 460 cGy, 465 cGy, 470cGy, 475 cGy, 480 cGy, 485 cGy, 490 cGy, 495 cGy or at least 500 cGy.

Irradiation may be administered on the same day of solid-organtransplantation. In some cases, the plurality of irradiation doses maybe delivered over a period of time after organ transplantation. In somecases, the plurality of irradiation doses may be delivered over a periodof at least 0.1 days, 0.2 days, 0.3 days, 0.4 days, 0.5 days, 0.6 days,0.7 days, 0.8 days, 0.9 days, 1.0 days, 1.1 days, 1.2 days, 1.3 days,1.4 days, 1.5 days, 1.6 days, 1.7 days, 1.8 days, 1.9 days, 2.0 days,2.1 days, 2.2 days, 2.3 days, 2.4 days, 2.5 days, 2.6 days, 2.7 days,2.8 days, 2.9 days, 3.0 days, 3.1 days, 3.2 days, 3.3 days, 3.4 days,3.5 days, 3.6 days, 3.7 days, 3.8 days, 3.9 days, 4.0 days, 4.1 days,4.2 days, 4.3 days, 4.4 days, 4.5 days, 4.6 days, 4.7 days, 4.8 days,4.9 days, 5.0 days, 5.1 days, 5.2 days, 5.3 days, 5.4 days, 5.5 days,5.6 days, 5.7 days, 5.8 days, 5.9 days, 6.0 days, 6.1 days, 6.2 days,6.3 days, 6.4 days, 6.5 days, 6.6 days, 6.7 days, 6.8 days, 6.9 days,7.0 days, 7.1 days, 7.2 days, 7.3 days, 7.4 days, 7.5 days, 7.6 days,7.7 days, 7.8 days, 7.9 days, 8.0 days, 8.1 days, 8.2 days, 8.3 days,8.4 days, 8.5 days, 8.6 days, 8.7 days, 8.8 days, 8.9 days, 9.0 days,9.1 days, 9.2 days, 9.3 days, 9.4 days, 9.5 days, 9.6 days, 9.7 days,9.8 days, 9.9 days, 10 days, 10.5 days, 11 days, 11.5 days, 12 days,12.5 days, 13 days, 13.5 days, 14 days, 14.5 days, 15 days, 15.5 days,16 days, 16.5 days, 17 days, 17.5 days, 18 days, 18.5 days, 19 days, 20days, 20.5 days, 21 days, 21.5 days, 22 days, 22.5 days, 23 days, 23.5days, 24 days, 24.5 days, 25 days, 25.5 days, 26 days, 26.5 days, 27days, 27.5 days, 28 days, 28.5 days, 29 days, 30 days, 30.5 days, 31days, 31.5 days, 32 days, 32.5 days, 33 days, 33.5 days, 34 days, 34.5days, 35 days, 35.5 days, 36 days, 36.5 days, 37 days, 37.5 days, 38days, 38.5 days, 39 days, 40 days, 40.5 days, 41 days, 41.5 days, 42days, 42.5 days, 43 days, 43.5 days, 44 days, 44.5 days, 45 days, 45.5days, 46 days, 46.5 days, 47 days, 47.5 days, 48 days, 48.5 days, 49days or at least 50 days.

In some cases, the doses of irradiation are delivered on a regularinterval over the course of administration. In other cases, the doses ofirradiation are not delivered on a regular interval over the course ofadministration. For example, irradiation may be delivered to the thymusgland on days 1 through 4, and days 7 through 11 after transplantation.

The irradiation may be targeted to a particular location of therecipient's body. In some cases, the irradiation may be targeted to atissue, an organ, a region of the body or the whole body. In some cases,irradiation may be targeted to the lymph nodes, the spleen, or thethymus or any other area known to a person of skill in the art. In somecases, the irradiation may be targeted to the same location when atleast more than one dose of irradiation is delivered to the patient. Inother cases, the irradiation may be targeted a different location whenat least more than one dose of irradiation is delivered to the patient.

During non-myeloablative conditioning, recipients may be monitored forthe development of conditions associated with non-myeloablativeconditioning. Such diseases include neutropenia (e.g., granulocytes<2,000/mL), thrombocytopenia (e.g., platelets <60,000/mL) and secondaryinfections. In some cases, G-CSF (e.g., 10 μg/kg/day) may beadministered for neutropenia. In some cases, any standard treatmentknown to one of skill in the art may be administered forthrombocytopenia or any secondary infections.

In some cases, non-myeloablative conditioning may be temporarily stoppedif a recipient develops neutropenia, thrombocytopenia or any secondaryinfections. Non-myeloablative conditioning may be continued onceneutropenia, thrombocytopenia and or any secondary infections areresolved. In some cases, if the recipient has a white blood count below1,000 cells/mm³, the recipient may be treated with G-CSF (e.g., 10μg/kg/day) following non-myeloablative conditioning.

Immunosuppression and Graft Management

Following either HLA-matched or HLA-mismatched solid organtransplantation and administration of the engineered HLA-matched orHLA-mismatched hematopoietic cells, the recipient may receive animmunosuppressive regimen. The immunosuppressive regimen may have twophases, an induction phase and a maintenance phase. Induction andmaintenance phase strategies may use different medicines at dosesadjusted to achieve target therapeutic levels to enhance long termtransplant persistence in the recipient. In some cases, the inductionphase may begin perioperatively. In some cases, the induction phase maybegin immediately after transplantation. In some cases, the inductionphase may be both perioperative and immediately after transplantation.In some cases, the immunosuppressive regimen may continue as amaintenance therapy until the recipient achieves chimerism. For example,chimerism may be stable mixed chimerism as described herein.

In some cases, the immunosuppressive regimen may include one agent. Inother cases, the immunosuppressive regimen may include more than oneagent. For example, suitable agents for the immunosuppressive regimenmay include a calcineurin inhibitor and/or an adjuvant. In some cases,the primary immunosuppressive agents include calcineurin inhibitors,which combine with binding proteins to inhibit calcineurin activity. Insome cases, the calcineurin inhibitor may be tacrolimus, cyclosporine A,or any calcineurin inhibitor known to one of skill in the art and may beadministered to the recipient at a dose effective to provide targetedimmunosuppression as a calcineurin inhibitor.

In some cases, cyclosporine A may be withdrawn from the recipient aftera duration of less than one month, two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months or less than 24 months.

In some cases, cyclosporine A may be withdrawn from the recipient aftera duration of more than one month, two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months or more than 24 months.

In some cases, the dose of cyclosporine A may slowly be tapered if therecipient meets clinical criteria for lack of rejection and GVHD. Forexample, the total amount of the cyclosporine A administered may bereduced over time. In some cases, tapering of the cyclosporine A mayoccur for a duration of less than one month, two months, three months,four months, five months, six months, seven months, eight months, ninemonths, ten months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months or less than 24 months such that at the endof the tapering regime, the dose of the cyclosporine A is tapered tozero. In some cases, tapering of the cyclosporine A may occur for aduration of more than one month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months or more than 24 months such that at the end of thetapering regime, the dose of the cyclosporine A is tapered to zero.

In some cases, the cyclosporine A may be delivered by a single dose tothe recipient. In other cases, the recipient may receive more than onedose of cyclosporine A. For example, a recipient may receive at leastone dose of cyclosporine A, two doses of cyclosporine A, three doses ofcyclosporine A, four doses of cyclosporine A, five doses of cyclosporineA, six doses of cyclosporine A, seven doses of cyclosporine A, eightdoses of cyclosporine A, nine doses of cyclosporine A, 10 doses ofcyclosporine A, 11 doses of cyclosporine A, 12 doses of cyclosporine A,13 doses of cyclosporine A, 14 doses of cyclosporine A, 15 doses ofcyclosporine A, 16 doses of cyclosporine A, 17 doses of cyclosporine A,18 doses of cyclosporine A, 19 doses of cyclosporine A, or 20 doses ofcyclosporine A.

In some cases, a plurality of cyclosporine A doses may be delivered overa period of time after organ transplantation. In some cases, theplurality of cyclosporine A doses may be delivered over a period of atleast 0.1 days, 0.2 days, 0.3 days, 0.4 days, 0.5 days, 0.6 days, 0.7days, 0.8 days, 0.9 days, 1.0 days, 1.1 days, 1.2 days, 1.3 days, 1.4days, 1.5 days, 1.6 days, 1.7 days, 1.8 days, 1.9 days, 2.0 days, 2.1days, 2.2 days, 2.3 days, 2.4 days, 2.5 days, 2.6 days, 2.7 days, 2.8days, 2.9 days, 3.0 days, 3.1 days, 3.2 days, 3.3 days, 3.4 days, 3.5days, 3.6 days, 3.7 days, 3.8 days, 3.9 days, 4.0 days, 4.1 days, 4.2days, 4.3 days, 4.4 days, 4.5 days, 4.6 days, 4.7 days, 4.8 days, 4.9days, 5.0 days, 5.1 days, 5.2 days, 5.3 days, 5.4 days, 5.5 days, 5.6days, 5.7 days, 5.8 days, 5.9 days, 6.0 days, 6.1 days, 6.2 days, 6.3days, 6.4 days, 6.5 days, 6.6 days, 6.7 days, 6.8 days, 6.9 days, 7.0days, 7.1 days, 7.2 days, 7.3 days, 7.4 days, 7.5 days, 7.6 days, 7.7days, 7.8 days, 7.9 days, 8.0 days, 8.1 days, 8.2 days, 8.3 days, 8.4days, 8.5 days, 8.6 days, 8.7 days, 8.8 days, 8.9 days, 9.0 days, 9.1days, 9.2 days, 9.3 days, 9.4 days, 9.5 days, 9.6 days, 9.7 days, 9.8days, 9.9 days, 10 days, 10.5 days, 11 days, 11.5 days, 12 days, 12.5days, 13 days, 13.5 days, 14 days, 14.5 days, 15 days, 15.5 days, 16days, 16.5 days, 17 days, 17.5 days, 18 days, 18.5 days, 19 days or atleast 20 days.

In some cases, each dose of cyclosporine A may be at least 0.1 mg/kg,0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg,1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg,2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg,4.1 mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7mg/kg, 4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg,5.4 mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg, 6.0mg/kg, 6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg,6.7 mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3mg/kg, 7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg,8.0 mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6mg/kg, 8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2 mg/kg,9.3 mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9mg/kg, or at least 10 mg/kg.

In some cases, the amount of cyclosporine A administered to the patientmay be determined by the amount of the cyclosporine A in thebloodstream. For example, the cyclosporine A may be administered at adose to achieve a range of 0-40 mg, 5-50 mg, 10-60 mg, 15-65 mg, 20-70mg, 25-75 mg, 30-80 mg, 35-85 mg, 40-90 mg, 45-95 mg, 50-100 mg, 55-105mg, 60-110 mg, 65-115 mg, 70-120 mg, 75-125 mg, 80-130 mg, 85-135 mg,90-140 mg, 95-145 mg, 100-150 mg, 105-155 mg, 110-160 mg, 115-165 mg,120-170 mg, 125-175 mg, 130-180 mg, 135-185 mg, 140-190 mg, 145-195 mg,150-200 mg, 160-210 mg, 170-220 mg, 180-230 mg, 190-240 mg, 200-250 mg,210-260 mg, 220-270 mg, 230-280 mg, 240-290 mg, 250-300 mg, 260-310 mg,270-320 mg, 280-330 mg, 290-340 mg, 300-350 mg, 310-360 mg, 320-370 mg,330-380 mg, 340-390 mg, 350-400 mg, 360-410 mg, 370-420 mg, 380-430 mg,390-440 mg, 400-450 mg, 410-460 mg, 420-470 mg, 430-480 mg, 440-490 mg,450-500 mg, 46-510 mg, 470-520 mg, 480-530 mg, 490-540 mg, 500-550 mg,510-560 mg, 520-570 mg, 530-580 mg, 540-590 mg, 550-600 mg, 560-610 mg,570-620 mg, 580-630 mg, 590-640 mg, 600-650 mg, 610-660 mg, 620-670 mg,630-680 mg, 640-690 mg, 650-700 mg or more than 700 mg.

In some cases, tacrolimus may be withdrawn from the recipient after aduration of more than one month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months or more than 24 months. In some cases, the dose oftacrolimus may slowly be tapered providing the recipient meets clinicalcriteria for lack of rejection and GVHD. For example, the total amountof tacrolimus administered may be reduced over time. In some cases,tapering of tacrolimus may occur for a duration of less than one month,two months, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months or less than 24months such that at the end of the tapering regime, the dose oftacrolimus is tapered to zero.

In some cases, tacrolimus may be withdrawn from the recipient after aduration of less than one month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months or less than 24 months. In some cases, the dose oftacrolimus may slowly be tapered providing the recipient meets clinicalcriteria for lack of rejection and GVHD. For example, the total amountof tacrolimus administered may be reduced over time. In some cases,tapering of tacrolimus may occur for a duration of more than one month,two months, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months or more than 24months such that at the end of the tapering regime, the dose oftacrolimus is tapered to zero.

In some cases, tacrolimus may be delivered by a single to the recipient.In other cases, the recipient may receive more than one dose ofTacrolimus. For example, a recipient may receive at least one dose ofTacrolimus, two doses of Tacrolimus, three doses of Tacrolimus, fourdoses of Tacrolimus, five doses of Tacrolimus, six doses of Tacrolimus,seven doses of Tacrolimus, eight doses of Tacrolimus, nine doses ofTacrolimus, 10 doses of Tacrolimus, 11 doses of Tacrolimus, 12 doses ofTacrolimus, 13 doses of Tacrolimus, 14 doses of Tacrolimus, 15 doses ofTacrolimus, 16 doses of Tacrolimus, 17 doses of Tacrolimus, 18 doses ofTacrolimus, 19 doses of Tacrolimus, or at least 20 doses of Tacrolimus.

In some cases, a plurality of tacrolimus doses may be delivered over aperiod of time after organ transplantation. In some cases, the pluralityof tacrolimus doses may be delivered over a period of at least 0.1 days,0.2 days, 0.3 days, 0.4 days, 0.5 days, 0.6 days, 0.7 days, 0.8 days,0.9 days, 1.0 days, 1.1 days, 1.2 days, 1.3 days, 1.4 days, 1.5 days,1.6 days, 1.7 days, 1.8 days, 1.9 days, 2.0 days, 2.1 days, 2.2 days,2.3 days, 2.4 days, 2.5 days, 2.6 days, 2.7 days, 2.8 days, 2.9 days,3.0 days, 3.1 days, 3.2 days, 3.3 days, 3.4 days, 3.5 days, 3.6 days,3.7 days, 3.8 days, 3.9 days, 4.0 days, 4.1 days, 4.2 days, 4.3 days,4.4 days, 4.5 days, 4.6 days, 4.7 days, 4.8 days, 4.9 days, 5.0 days,5.1 days, 5.2 days, 5.3 days, 5.4 days, 5.5 days, 5.6 days, 5.7 days,5.8 days, 5.9 days, 6.0 days, 6.1 days, 6.2 days, 6.3 days, 6.4 days,6.5 days, 6.6 days, 6.7 days, 6.8 days, 6.9 days, 7.0 days, 7.1 days,7.2 days, 7.3 days, 7.4 days, 7.5 days, 7.6 days, 7.7 days, 7.8 days,7.9 days, 8.0 days, 8.1 days, 8.2 days, 8.3 days, 8.4 days, 8.5 days,8.6 days, 8.7 days, 8.8 days, 8.9 days, 9.0 days, 9.1 days, 9.2 days,9.3 days, 9.4 days, 9.5 days, 9.6 days, 9.7 days, 9.8 days, 9.9 days, 10days, 10.5 days, 11 days, 11.5 days, 12 days, 12.5 days, 13 days, 13.5days, 14 days, 14.5 days, 15 days, 15.5 days, 16 days, 16.5 days, 17days, 17.5 days, 18 days, 18.5 days, 19 days or at least 20 days.

In some cases, each dose of tacrolimus may be at least 0.1 mg/kg, 0.2mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg,0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg,2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg,3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg,4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg, 5.4mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg, 6.0 mg/kg,6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg,7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg, 8.0mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6 mg/kg,8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg,or at least 10 mg/kg.

In some cases, the amount of tacrolimus administered to the patient isdetermined by the amount of tacrolimus in the bloodstream. For example,tacrolimus may be administered at a dose to achieve a range of 0-40 mg,5-50 mg, 10-60 mg, 15-65 mg, 20-70 mg, 25-75 mg, 30-80 mg, 35-85 mg,40-90 mg, 45-95 mg, 50-100 mg, 55-105 mg, 60-110 mg, 65-115 mg, 70-120mg, 75-125 mg, 80-130 mg, 85-135 mg, 90-140 mg, 95-145 mg, 100-150 mg,105-155 mg, 110-160 mg, 115-165 mg, 120-170 mg, 125-175 mg, 130-180 mg,135-185 mg, 140-190 mg, 145-195 mg, 150-200 mg, 160-210 mg, 170-220 mg,180-230 mg, 190-240 mg, 200-250 mg, 210-260 mg, 220-270 mg, 230-280 mg,240-290 mg, 250-300 mg, 260-310 mg, 270-320 mg, 280-330 mg, 290-340 mg,300-350 mg, 310-360 mg, 320-370 mg, 330-380 mg, 340-390 mg, 350-400 mg,360-410 mg, 370-420 mg, 380-430 mg, 390-440 mg, 400-450 mg, 410-460 mg,420-470 mg, 430-480 mg, 440-490 mg, 450-500 mg, 46-510 mg, 470-520 mg,480-530 mg, 490-540 mg, 500-550 mg, 510-560 mg, 520-570 mg, 530-580 mg,540-590 mg, 550-600 mg, 560-610 mg, 570-620 mg, 580-630 mg, 590-640 mg,600-650 mg, 610-660 mg, 620-670 mg, 630-680 mg, 640-690 mg, 650-700 mgor more than 700 mg.

The levels of either cyclosporine or tacrolimus in the recipient may bemonitored. At the onset of immunosuppression, the levels of eithercyclosporine or tacrolimus may be, for example, in the range of 0-15ng/mL, 5-15 ng/mL, 10-20 ng/mL, 15-25 ng/mL, 20-30 ng/mL, 25-35 ng/mL,30-40 ng/mL, 35-45 ng/mL or 40-50 ng/mL in the recipient. In some cases,the levels of either cyclosporine or tacrolimus may be reduced after aperiod of time in the recipient. For example, the period of time may beless than one week, two weeks, three weeks, four weeks, five weeks, sixweeks, seven weeks, eight weeks, nine weeks, ten weeks, 11 weeks, 12weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26weeks, 27 weeks, 28 weeks, 29 weeks or less than 33 weeks. In somecases, the levels of either cyclosporine or tacrolimus may be reduced towithin the range of 0-1 ng/mL, 0.5-1.5 ng/mL, 1.0-2.0 ng/mL, 1.5-2.5ng/mL, 2.0-3.0 ng/mL, 2.5-3.5 ng/mL, 3.0-4.0 ng/mL, 3.5-4.5 ng/mL,4.0-5.0 ng/mL, 5.5-6.5 ng/mL, 6.0-7.0 ng/mL, 6.5-7.5 ng/mL, 7.0-8.0ng/mL, 8.5-9.5 ng/mL or 9.0-10.0 ng/mL in the recipient.

In some cases, a calcineurin inhibitor may be administered to therecipient in combination with an inhibitor of purine metabolism (e.g.,mycophenolate mofetil). For example, cyclosporine A and mycophenolatemofetil may be used in the case of kidney transplantation.

In some cases, the adjuvant may be withdrawn from the recipient after aduration of more than one month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months or more than 24 months. In some cases, the dose of theadjuvant may slowly be tapered providing the recipient meets clinicalcriteria for lack of rejection and GVHD. For example, the total amountof the adjuvant administered may be reduced over time. In some cases,tapering of the adjuvant may occur for a duration of more than onemonth, two months, three months, four months, five months, six months,seven months, eight months, nine months, ten months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months or morethan 24 months such that at the end of the tapering regime, the dose ofthe purine metabolism inhibitor is tapered to zero.

In some cases, the adjuvant may be withdrawn from the recipient after aduration of less than one month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months or less than 24 months. In some cases, the dose of theadjuvant may slowly be tapered providing the recipient meets clinicalcriteria for lack of rejection and GVHD. For example, the total amountof the adjuvant administered may be reduced over time. In some cases,tapering of the adjuvant may occur for a duration of less than onemonth, two months, three months, four months, five months, six months,seven months, eight months, nine months, ten months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months or lessthan 24 months such that at the end of the tapering regime, the dose ofthe purine metabolism inhibitor is tapered to zero.

Adjuvant agents may be used to enhance immunosuppression whiledecreasing the dose and toxicity of other individual agents that arepart of the immunosuppressive regimen. In some cases, adjuvant agentsmay be combined with a calcineurin inhibitor. For example, adjuvantagents may include steroids, azathioprine, mycophenolate mofetil,sirolimus, an antibody or any adjuvant agent known to one of skill inthe art and may be administered to the recipient at a dose effective toenhance immunosuppression.

In some cases, antibody-based therapy may be used to avoid or reduce thedose of calcineurin inhibitors in the immunosuppressive regimen. Forexample, antibody-based therapy may include monoclonal (e.g.,muromonab-CD3) antibodies, polyclonal antibodies and/or anti-CD25antibodies (eg, basiliximab, daclizumab). In some cases, antibody-basedtherapy may be administered during the early post-transplant period. Forexample, early post-transplant may be up to 8 weeks following thetransplant.

Graft management may include preventing, inhibiting or suppressing acuterejection with immunosuppressive drugs. In some cases, multiple agentsmay be used to prevent, inhibit or suppress episodes of acute rejection.For example, an agent may be a steroid. In some cases, one or more thanone steroid may be used to prevent, inhibit or suppress episodes ofacute rejection. Any steroid known to one of skill in the art suitablefor preventing, inhibiting or suppressing acute rejection may be used.For example, any dose, mode of administration and duration ofadministration for any steroid known to one of skill in the art suitablefor preventing, inhibiting or suppressing acute rejection may be used.In some cases, administration of the steroid may be tapered to amaintenance dose.

For example, an agent may be antithymocyte globulin. In some cases,antithymocyte globulin may be used to prevent, inhibit or suppressepisodes of acute rejection. Any dose, mode of administration andduration of administration for antithymocyte globulin suitable forpreventing, inhibiting or suppressing acute rejection may be used. Insome cases, administration of antithymocyte globulin may be tapered to amaintenance dose.

For example, an agent may be muromonab-CD3. In some cases, muromonab-CD3may be used to prevent, inhibit or suppress episodes of acute rejection.Any dose, mode of administration and duration of administration formuromonab-CD3 suitable for preventing, inhibiting or suppressing acuterejection may be used. In some cases, administration of muromonab-CD3may be tapered to a maintenance dose.

Chimerism

Following either HLA-matched or HLA-mismatched solid organtransplantation and administration of the engineered HLA-matched orHLA-mismatched hematopoietic cells, the recipient may be monitored forchimerism. Recipients who exhibit greater than 95% donor cells in agiven blood cell lineage by any analysis to determine chimerism at anytime post-transplantation may be classified as having full donorchimerism. In some cases, mixed chimerism may be greater than 1%donor-derived cells of a given lineage but less than 95% donor-derivedDNA.

The relative dose of CD3+ cells and CD34+ cells, and the total dose ofthese cells, can influence whether an individual develops stable mixedchimerism. In some cases, for example where the CD3+ cells and the CD34+cells are HLA matched between donor and recipient, the dose of CD3+cells can be at least about 1×10⁶ cells/kg and the dose of CD34+ cellscan be at least about 0.5×10⁶/kg. In some cases, where the CD3+ cellsand the CD34+ cells are HLA mismatched between donor and recipient, thedose of CD3+ cells can be at least about 10×10⁶ cells/kg and may be lessthan 50×10⁷ cells/kg, and the dose of CD34+ cells can be at least about1×10⁶/kg, at least about 5×10⁶/kg, at least about 10×10⁶/kg, at least15×10⁶/kg or more. Where the dose of CD34+ cells is less than 15×10⁶/kgit may be desirable to administer a higher dose of CD3+ cells, e.g. upto about 25×10⁶/kg, up to about 30×10⁶/kg, up to about 35×10⁶/kg, up toabout 40×10⁶/kg, up to about 45×10⁶/kg, up to about 50×10⁶/kg.

HLA matched patients there needs to be at least injected, but in thecase of HLA mismatched there needs to be at least 10×10{circumflex over( )}6 T cells/kg injected along with CD34 cells to achieve persistentmixed chimerism for at least 6 months. If the CD34 cell dose is below15×10{circumflex over ( )}6 cells/kg in mismatched patients, then atleast 50×10{circumflex over ( )}6 T cells/kg is needed.

Individuals who exhibit mixed chimerism may be further classifiedaccording to the evolution of chimerism, where improving mixed chimerismmay be a continuous increase in the proportion of donor cells over aperiod of time (e.g., at least a 6-months). In some cases, stable mixedchimerism may include fluctuations in the percentage of recipient cellsover time, without complete loss of donor cells.

The doses of CD34+ and CD3+ cells used in the engineered hematopoieticcell composition are selected in order to achieve stable mixed chimerismin the recipient after transplant. Mixed chimerism is defined as greaterthan 1% recipient DNA. In some cases, mixed chimerism may include apercentage of cells derived from the donor and a percentage of cellsderived from the recipient. In some cases, mixed chimerism is more than70% of the cells in the recipient being derived from the donor. In othercases, mixed chimerism is more than 10% of the cells in the recipientbeing derived from the donor, more than 15% of the cells in therecipient being derived from the donor, more than 20% of the cells inthe recipient being derived from the donor, more than 25% of the cellsin the recipient being derived from the donor, more than 30% of thecells in the recipient being derived from the donor, more than 35% ofthe cells in the recipient being derived from the donor, more than 40%of the cells in the recipient being derived from the donor, more than45% of the cells in the recipient being derived from the donor, morethan 50% of the cells in the recipient being derived from the donor,more than 55% of the cells in the recipient being derived from thedonor, more than 56% of the cells in the recipient being derived fromthe donor, more than 57% of the cells in the recipient being derivedfrom the donor, more than 58% of the cells in the recipient beingderived from the donor, more than 59% of the cells in the recipientbeing derived from the donor, more than 60% of the cells in therecipient being derived from the donor, more than 61% of the cells inthe recipient being derived from the donor, more than 62% of the cellsin the recipient being derived from the donor, more than 63% of thecells in the recipient being derived from the donor, more than 64% ofthe cells in the recipient being derived from the donor, more than 65%of the cells in the recipient being derived from the donor, more than66% of the cells in the recipient being derived from the donor, morethan 67% of the cells in the recipient being derived from the donor,more than 68% of the cells in the recipient being derived from thedonor, more than 69% of the cells in the recipient being derived fromthe donor, more than 70% of the cells in the recipient being derivedfrom the donor, more than 71% of the cells in the recipient beingderived from the donor, more than 72% of the cells in the recipientbeing derived from the donor, more than 73% of the cells in therecipient being derived from the donor, more than 74% of the cells inthe recipient being derived from the donor, more than 75% of the cellsin the recipient being derived from the donor, more than 76% of thecells in the recipient being derived from the donor, more than 77% ofthe cells in the recipient being derived from the donor, more than 78%of the cells in the recipient being derived from the donor, more than79% of the cells in the recipient being derived from the donor, morethan 80% of the cells in the recipient being derived from the donor,more than 81% of the cells in the recipient being derived from thedonor, more than 82% of the cells in the recipient being derived fromthe donor, more than 83% of the cells in the recipient being derivedfrom the donor, more than 84% of the cells in the recipient beingderived from the donor, more than 85% of the cells in the recipientbeing derived from the donor, more than 86% of the cells in therecipient being derived from the donor, more than 87% of the cells inthe recipient being derived from the donor, more than 88% of the cellsin the recipient being derived from the donor, more than 89% of thecells in the recipient being derived from the donor, more than 90% ofthe cells in the recipient being derived from the donor, more than 91%of the cells in the recipient being derived from the donor, more than92% of the cells in the recipient being derived from the donor, morethan 93% of the cells in the recipient being derived from the donor,more than 94% of the cells in the recipient being derived from thedonor, more than 95% of the cells in the recipient being derived fromthe donor, more than 96% of the cells in the recipient being derivedfrom the donor, more than 97% of the cells in the recipient beingderived from the donor, more than 98% of the cells in the recipientbeing derived from the donor ore more than 99% of the cells in therecipient being derived from the donor.

Mixed chimerism may be stable following a transplant using theCD34+/CD3+ engineered hematopoietic cell composition described herein.In some cases, mixed chimerism is stable. In some cases, stable mixedchimerism lasts for at least 6 months after treatment with anyengineered hematopoietic cell composition of CD34+/CD3+ cells describedherein. In some cases, stable mixed chimerism may persist for more thanfive days, more than 10 days, more than 15 days, more than 20 days, morethan 25 days, more than 30 days, more than 35 days, more than 40 days,more than 45 days, more than 50 days, more than 55 days, more than 60days, more than 65 days, more than 70 days, more than 75 days, more than80 days, more than 85 days, more than 90 days, more than 95 days, morethan 100 days, more than 105 days, more than 110 days, more than 115days, more than 120 days, more than 125 days, more than 130 days, morethan 135 days, more than 140 days, more than 145 days, more than 150days, more than 155 days, more than 160 days, more than 165 days, morethan 170 days, more than 175 days, more than 180 days, more than 185days, more than 190 days, more than 195 days, more than 200 days, morethan 205 days, more than 210 days, more than 215 days, more than 220days, more than 225 days, more than 230 days, more than 235 days, morethan 240 days, more than 245 days, more than 250 days, more than 255days, more than 260 days, more than 265 days, more than 270 days, morethan 275 days, more than 280 days, more than 285 days, more than 290days, more than 295 days, more than 300 days, more than 305 days, morethan 310 days, more than 315 days, more than 320 days, more than 325days, more than 330 days, more than 335 days, more than 340 days, morethan 345 days, more than 350 days, more than 355 days, more than 360days, more than 365 days, more than 370 days, more than 375 days, morethan 380 days, more than 385 days, more than 390 days, more than 395days, more than 400 days, more than 405 days, more than 410 days, morethan 415 days, more than 420 days, more than 425 days, more than 430days, more than 435 days, more than 440 days, more than 445 days, morethan 450 days, more than 455 days, more than 460 days, more than 465days, more than 470 days, more than 475 days, more than 480 days, morethan 485 days, more than 490 days, more than 495 days, or more than 500days.

Mixed chimerism may be determined by measuring the percentage of donorcells for a single cell type within the recipient. For example, mixedchimerism may be determined by the percentage of donor-derivedgranulocytes in the recipient. In some cases, mixed chimerism may bedetermined by measuring the percentage of donor cells for a plurality ofcell types within the recipient. For example, mixed chimerism may bedetermined by the percentage of donor-derived granulocytes, naturalkiller cells, B cells and T cells in the recipient.

In some cases, the percentage of donor-derived granulocytes in therecipient may be measured. In some cases, the percentage ofdonor-derived granulocytes may be constant in the recipient aftertransplantation. In other cases, the percentage of donor-derivedgranulocytes may not be constant in the recipient after transplantation.In other cases, the percentage of donor-derived granulocytes changesover time in the recipient after transplantation. For example, over aperiod of 60 days after transplantation, the percentage of donor-derivedgranulocytes in the recipient may be at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 20%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

In some cases, the percentage of donor-derived natural killer cells inthe recipient may be measured. In some cases, the percentage ofdonor-derived natural killer cells may be constant in the recipientafter transplantation. In other cases, the percentage of donor-derivednatural killer cells may not be constant in the recipient aftertransplantation. In other cases, the percentage of donor-derived naturalkiller cells changes over time in the recipient after transplantation.For example, over a period of 60 days after transplantation, thepercentage of donor-derived natural killer cells in the recipient may beat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 20%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% orat least 99%.

In some cases, the percentage of donor-derived B cells in the recipientmay be measured. In some cases, the percentage of donor-derived B cellsmay be constant in the recipient after transplantation. In other cases,the percentage of donor-derived B cells may not be constant in therecipient after transplantation. In other cases, the percentage ofdonor-derived B cells change over time in the recipient aftertransplantation. For example, over a period of 60 days aftertransplantation, the percentage of donor-derived B cells in therecipient may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 20%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or at least 99%.

In some cases, the percentage of donor-derived T cells in the recipientmay be measured. In some cases, the percentage of donor-derived T cellsmay Te constant in the recipient after transplantation. In other cases,the percentage of donor-derived T cells may not be constant in therecipient after transplantation. In other cases, the percentage ofdonor-derived T cells change over time in the recipient aftertransplantation. For example, over a period of 60 days aftertransplantation, the percentage of donor-derived T cells in therecipient may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 20%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or at least 99%.

There are a plurality of methods of testing for chimerism that arereadily available and known to those of skill in the art. Any method oftesting for chimerism that distinguishes donor or recipient origin of acell is suitable for use in the methods described herein.

In some cases, the methods of testing for chimerism may include geneticbased methods. For example, polymerase chain reaction (PCR) basedmethods which utilize probes may be used. In some cases, probes for PCRbased methods may be probes for microsatellite analysis. For anotherexample, commercial kits that distinguish polymorphisms in shortterminal repeat lengths of donor and host origin are readily availableand known to those of skill in the art.

In some cases, major histocompatibility complex (MHC) typing may be usedfor testing chimerism. For example, MHC typing may be used to test thetype of cells in the blood. In some cases, MHC typing may be used incombination with flow cytometry. In some case, an analysis ofHLA-stained cells by flow cytometry may be performed.

The methods described herein are provided such that a recipient mayachieve stable mixed chimerism sufficient to allow withdrawal ofimmunosuppressive drugs. For example, withdrawal of immunosuppressivedrugs may include tapering immunosuppressive drugs. In other cases,withdrawal of immunosuppressive drugs may include immediate withdrawalof immunosuppressive drugs. In some cases, mixed chimerism persists forat least six months prior to withdrawal of immunosuppressive drugs. Inother cases, mixed chimerism persists for at least one month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months or at least 24months. In some cases, the dose of the adjuvant may slowly be taperedproviding the recipient meets clinical criteria for lack of rejectionand GVHD. For example, the total amount of the adjuvant administered maybe reduced over time. In some cases, tapering of the adjuvant may occurfor a duration of at least one month, two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months or at least 24 months.

In some cases, a lack of rejection episodes may coincide with mixedchimerism prior to withdrawal of immunosuppressive drugs. In some cases,a lack of rejection episodes may be consistent for at least six monthsprior to withdrawal of immunosuppressive drugs. In other cases, a lackof rejection episodes may be consistent for at least one month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months or at least 24months. In some cases, the dose of the adjuvant may slowly be taperedproviding the recipient meets clinical criteria for lack of rejectionand GVHD. For example, the total amount of the adjuvant administered maybe reduced over time. In some cases, tapering of the adjuvant may occurfor a duration of at least one month, two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months or at least 24 months.

In some cases, a lack of GVHD and lack of rejection episodes coincideswith mixed chimerism prior to withdrawal of immunosuppressive drugs. Insome cases, a lack of GVHD and lack of rejection episodes may beconsistent for at least six months prior to withdrawal ofimmunosuppressive drugs. In other cases, a lack of GVHD and lack ofrejection episodes may be consistent for at least one month, two months,three months, four months, five months, six months, seven months, eightmonths, nine months, ten months, 11 months, 12 months, 13 months, 14months, 15 months, 16 months, 17 months, 18 months, 19 months, 20months, 21 months, 22 months, 23 months or at least 24 months. In somecases, the dose of the adjuvant may slowly be tapered providing therecipient meets clinical criteria for lack of rejection and GVHD. Forexample, the total amount of the adjuvant administered may be reducedover time. In some cases, tapering of the adjuvant may occur for aduration of at least one month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months or at least 24 months.

In order to determine if tapering of the immunosuppressive regimen isappropriate for the recipient, the recipient may be tested for mixedchimerism, usually at regular intervals. For example, regular intervalsmay be monthly, semi-monthly, weekly, bi-monthly, annually, bi-annuallyor the like.

The invention now being fully described, it is apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXAMPLES

The present disclosure has been described in terms of particular casesfound or proposed to comprise preferred modes for the practice of thedisclosure. It is appreciated by those of skill in the art that, inlight of the present disclosure, numerous modifications and changes canbe made in the particular embodiments exemplified without departing fromthe intended scope of the disclosure. For example, due to codonredundancy, changes can be made in the underlying DNA sequence withoutaffecting the protein sequence. Moreover, due to biological functionalequivalency considerations, changes can be made in protein structurewithout affecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

For further elaboration of general techniques useful in the practice ofthis disclosure, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. With respect totissue culture and embryonic stem cells, the reader may wish to refer toTeratocarcinomas and embryonic stem cells: A practical approach (E. J.Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in MouseDevelopment (P. M. Wasserman et al. eds., Academic Press 1993);Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth.Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells:Prospects for Application to Human Biology and Gene Therapy (P. D.Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present disclosure and are not intended to limit thescope of what is regarded as the disclosure nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1—TLI and ATG Conditioning for Combined Kidney and Blood StemCell Transplantation

Immune tolerance to HLA haplotype matched living related donor kidneyallografts is developed in order to remove the requirement for thelifelong use of immunosuppressive drugs and to improve the long termgraft survival. Currently, these haplotype matched recipients accountfor about half of living related donor kidney transplants performed atmost medical centers in the United States. During the past 10 years anincreasing proportion of grafts in most centers were from living relateddonors as an alternative to cadaver grafts. Although the living relateddonor transplants have improved survival as compared to cadavertransplants, about 40 to 50% of the living donor grafts are still lostwithin about 10 years. In addition, the recipients usually receive amixture of 3 maintenance immunosuppressive drugs including a calcineurininhibitor, prednisone, and mycophenolate mofetil. The latter drugs haveside effects that include hypertension, nephrotoxicity, and heartdisease that contribute to long term patient disability and graft loss.

Our preclinical studies showed that conditioning with TLI and ATG isadvantageous for inducing tolerance after combined organ and bone marrowtransplantation because the conditioning regimen prevents GVHD ascompared to TBI. Approximately 1,000 fold more donor T cells are neededto induce lethal GVHD using TLI/ATG as compared to TBI conditioning. Thebasis of protection against GVHD is the change in the balance ofresidual host T cells that favors the host natural killer (NK) T cellsubset. The latter cells become the predominant T cell subset in TLI/ATGconditioned mice and produce large amounts of IL-4 that polarizeconventional donor T cells toward a Th2 bias, thereby attenuating GVHD.Several laboratories have shown that NK T cells that survive in vivoirradiation are themselves polarized toward a Th2 bias.

Based on the protective effect of TLI/ATG against GVHD in thepreclinical studies, this conditioning regimen has been successfullytested as a non-myeloablative regimen for HLA matched celltransplantation for patients. Modifications were made in theconditioning regimen and make-up of the hematopoetic cell graft for theadaptation. The hematopoietic cell graft was “engineered” to contain adefined number of purified CD34+ hematopoietic progenitor cells selectedby immunomagnetic bead columns and a defined number of donor T cells.Other aspects of the conditioning regimen remained the same, includingthe TLI and ATG schedule. A post-transplant immunosuppressive regimen of1 month of MMF and 6 months of cyclosporine was adapted from standardprotocols for patients receiving hematopoietic cell transplants to treatleukemia and lymphoma. Of note, the conditioning regimen was performedwith the start of transplant surgery on day 0, and infusion of donorhematopoietic cells was administered on day 11. This allowed for thefurther adaptation of the protocol to cadaver transplants in the future,and was the schedule used in the numerous preclinical studies ofcombined transplantation using TLI/ATG.

Twenty patients were given combined HLA matched grafts, followed up frombetween 5 to 86 months. Nineteen of these became chimeras without thedevelopment of GVHD. Seventeen developed persistent chimerism (>6months), and of these 14 were withdrawn from immunosuppressive drugs, 2of the 17 patients are undergoing drug tapering, and 4 of 20 failed drugwithdrawal (either due to failure to maintain chimerism for >6 months,or due to rejection episodes). All 20 patients currently had excellentgraft function at the last observation point, and were discharged about5 hospital days after transplant surgery. The patients needed 3-16 CD34+cell/kg and 1×106 CD3+ cell/Kg.

In order to apply the tolerance protocol described above to HLAmismatched patients, adjustments in the doses of CD34+ and CD3+ donorcells were made based on preliminary studies of 17 HLA halotypemismatched patients who were given the TLI and ATG conditioning regimenand a donor cell transplant to treat leukemia or lymphoma. The dose ofCD34+ cells was gradually escalated to ≥10×106 cell/Kg and the CD3+ dosewas gradually escalated to 10×106 cells/kg. The majority of patientsgiven the latter cell dose achieved mixed chimerism whereas none of thepatients given cell doses lower than these levels achieved mixedchimerism. Accordingly, a dose escalation clinical study was performedon 4 HLA haplotype matched patients given combined kidney andhematopoietic cell transplants with the goal of achieving target dosesof ≥10×106/Kg CD34+ cells and ≥10×106/Kg CD3+ cells. The purpose of thestudy was to determine whether the achievement of these target doseswould result in the development of mixed chimerism for >6 months and theability to withdraw immunosuppressive drugs thereafter. Some details ofthe clinical protocol are given below.

The gene array testing indicates that there is a change in the pre topost-transplant gene expression profiles in patients who met drugwithdrawal criteria such that their profiles became considerably bettermatched to those of the rare “operationally” tolerant patients whostopped conventional immunosuppressive drugs and did not develop graftrejection at their first monitoring time point. This was not the casewith the patients who failed to meet withdrawal criteria. Thedifferences in gene expression pattern between the 2 groups decreased atlater time points. It should be noted that the “operationally” tolerantpatients were all HLA mismatched. This indicates that the tolerant geneexpression profiles can be used to predict the tolerant state in bothmatched and mismatched patients.

Clinical Protocol

This was a single-center, open-label study in adult renal transplantpatients. Patients received TLI, RATG and an infusion of G-CSF“mobilized blood” mononuclear cells that had been enriched for CD34+cells and contained a defined number of CD34+ and CD3+ donor cells.Immunosuppressive drugs consisted of 9 months of mycophenolate mofetil(MMF; 15 mg/Kg twice per day starting on day 10), and a tapering courseof daily tacrolimus starting on day 0 that is discontinued at a targetof 12 months. The immunosuppressive drug combination of a calcineurininhibitor and a purine metabolism inhibitor was similar to that usedpreviously. At serial time points (1) graft function was monitored, (2)chimerism was measured in recipient white blood cell subsets and (3)protocol biopsies of the graft is obtained. If chimerism failed todevelop or is lost during the first six months, or if a rejectionepisode or GVHD occurred or if there was histological evidence ofrejection in graft biopsies then tacrolimus and/or MMF was notwithdrawn, and the patient would be followed thereafter as a treatmentfailure. Recipients in the study were given a target dose of ≥10×106CD34+ cells/Kg and an escalating dose of T cells to achieve the targetdose of 1×107/Kg T cells from the “mobilized” peripheral bloodmononuclear cells harvested from the donor. Of the 4 patients enrolledin the protocol, 2 did not receive the target cell doses and 2 did(Table 1).

Study Therapies. During the course of this study, patients receive 5intravenous injections of rabbit ATG (Thymoglobulin), a total of 1,200cGy of total lymphoid irradiation, a single infusion of donor cells,transient immunosuppression (MMF and Tacrolimus), and prophylacticanti-viral, anti-fungal and antibacterial agents.

Patients receive a total of 5 intravenous doses of Thymoglobulin over a5 day period; each dose is 1.5 mg/kg. Thymoglobulin is administered onthe day of transplantation (intra-operatively before the transplantedorgan is perfused with host blood) and on the subsequent 4 dayspost-transplant.

Patients receive ten treatments of fractionated irradiation (120 cGyeach) targeted to the lymph nodes, spleen and thymus gland on days 1through 4, and days 7 through 11, after transplantation such that thetotal dose of TLI is 1,200 cGy. Two doses are given on day 10 or 11 toachieve a total of 10 doses. TLI is given to the inverted Y and mantlefields. During the administration of TLI, patients are monitored for thedevelopment of neutropenia (granulocytes <2,000/mL), thrombocytopenia(platelets <60,000/mL) and infection. TLI is withheld for any of theseproblems, and G-CSF (10 μg/kg/day) is given for neutropenia. TLI isreinstated once neutropenia and/or thrombocytopenia resolves. At thecompletion of TLI, all patients are given G-CSF (10 μg/kg/day) if thewhite blood cell count is below 1,000 cells/mm3. TLI is completed by day11 if no doses are withheld.

A single intravenous infusion of cryopreserved HLA-haplotype matchedliving related donor, G-CSF mobilized blood mononuclear cells (recoveredfrom donor peripheral blood using apheresis), that has been “engineered”is administered to patients on the day of completion of TLI. Harvestingof donor cells is performed in the following fashion: Approximately 6weeks before renal transplantation, the donor is given G-CSF daily (16mg/kg/day) for five days, and mononuclear cells are harvested by anapheresis of up to 20 liters according to procedures previously approvedby the Stanford Committee on Medical Human Subjects for HLA-haplotypematched peripheral blood stem cell (PBSC) transplantation. In addition,a second session of up to 12 liters may be carried out for optimal cellrecovery. Cells are selected for CD34+ cells on Isolex columns, Columnflow through is collected also. Both CD34+ cells and flow through cellsare cryopreserved and thawed according to standard procedures at theStanford Blood and Marrow Transplantation laboratory. The target dose ofCD34+ cells to be injected is ≥10×106 cells/kg. A defined target dose of(1×107/kg CD3+) T cells is administered by injecting column flow throughcells along with enriched CD34+ cells intravenously. The dose of flowthrough cells are calculated based on the content of CD3+ cellsdetermined by immunofluorescent staining. This dose was used in the 17haplotype matched patients with leukemia and lymphoma. The majority ofthese patients developed persistent mixed chimerism, and none developedacute GVHD. The dose of T cells is increased if the first 3 recipientsof combined transplants fail to achieve persistent chimerism, and isdecreased if the first 3 recipients develop complete chimerism or if anypatient develops GVHD.

Corticosteroid therapy was limited to 60-120 mg Solumedrol (I.V.) aspremedication on the days of ATG infusions to reduce ATG side effects.After the last dose of ATG, a tapering course of prednisone starting at30 mg/d and reducing by 5 mg/d is given until day 10.

MMF therapy commenced on the day of the donor cell infusion (day 10) at15 mg/Kg twice per day. MMF therapy was maintained for 6 months, andthen tapered and stopped at 9 months.

Tacrolimus was started on day 0, adjusted to achieve a standing wholeblood trough level. As long as the criteria for immunosuppressive drugtapering are met, Tacrolimus was tapered beginning at month 9, andstopped by month 12.

Criteria for continued tapering of immunosuppressive drugs (Tacrolimus)through month 12 and MMF through month 9 were as follows: 1) Sustainedchimerism for at least 6 months; 2) No clinical rejection episodes; 3)Protocol biopsies show no evidence of acute or chronic rejection; 4) NoGVHD. Patients who do not meet these criteria are considered treatmentfailures, and further tapering of drugs is not performed.

If acute or chronic GVHD is observed that would ordinarily be treatedwith immunosuppressive drugs, then the patient is considered a treatmentfailure. Immunosuppressive drugs are administered according to standardpractice.

Rejection episodes are treated with standard anti-rejection therapywhich includes the use of intravenous methyl prednisolone and thepatient is considered a treatment failure. If no response to two coursesof steroids is found, then a course of anti-lymphocyte antibody isgiven. Tacrolimus is given at conventional doses during rejectionepisodes. Once a rejection episode occurs, patients will return toconventional doses of maintenance immunosuppressive drugs and no furthertapering is attempted as per the protocol above. Currently 95% of acuterejection episodes are reversed.

Since the initial course of TLI and ATG is expected to induce a markeddepletion of T cells, there is an increased risk of new or recrudescentviral infection, including cytomegalovirus (CMV), Epstein-Barr virus,Herpes zoster and Herpes simplex viruses as compared to conventionalimmunosuppressive protocols. Anti-viral prophylaxis for CMV is given asfollows: Valganciclovir (900 mg/d:P.O.) was given for the first 14 daysadjusted for renal function or during the first 14 days, ganciclovir(DHPG), 5 mg/kg, is given IV adjusted for renal function. After the14-day course all protocol transplant recipients were placed on thevalganciclovir (900 mg/d) adjusted for renal function. This wascontinued for a minimum of 90 days and if the absolute lymphocyte countis under 400, is continued until the time of steroid discontinuation.

Bactrim (1 single strength tablet per day) was given orally for one yearfor prophylaxis of Pneumocystis carinii pneumonia (PCP) and Mycostatinmouthwashes daily for three weeks for candida prophylaxis. Standardperi-operative antibiotics will include Ancef (1 mg, i.v., 3 doses at8-hour intervals) and Gentamicin (1.7 mg/kg, i.v., one dose at the timeof transplant). Antibacterial agents are subject to appropriatesubstitution according to patient allergies.

A surveillance biopsy is performed just before all immunosuppressivedrugs are stopped posttransplant. In addition, “for-cause” biopsies areobtained within 48 hours of an unexplained or unresolved 20% increase inserum creatinine.

Of the two patients who failed to receive the targeted cell doses,patient #1 failed to have mixed chimerism persist for more than 1 month.This patient received 3×106 CD3+ cells/Kg as part of the dose escalationstudy, but did receive the targeted number of CD34+ cells/Kg. Thispatient was not withdrawn from immunosuppressive drugs, and remains onTacrolimus and MMF with good graft function.

Patients #2 and #3 received the targeted cell dose of CD3+ T cells(10×106 cells/Kg) and the targeted dose of CD34+ cells. Both patientsdeveloped persistent mixed chimerism for 10 months and 5 monthsrespectively at present, and meet drug withdrawal criteria. The patternof persistent mixed chimerism is shorter for patient #3 in FIG. 2.

Patient #4 received 10×106 CD3+ cells/Kg but did not meet the requisitenumber of CD34+ cells due to poor mobilization of the donor CD34+ cellsdespite two courses of G-CSF administered to the donor. This patientfailed to develop chimerism, and remains on immunosuppressive drugs withgood graft function.

Example 2. Effect of T Cell Dose on Development of Mixed Chimerism

Patients were transplanted as described in Example 1. The HLA-matchingcharacteristics, conditioning regiment and donor cell composition wereperformed according to Table 1.

TABLE 1 Serum Total CD34⁺ CD3⁺ Creatinine Chimerism Patient # Dose CellCell at last Duration (>30% months Age/ ESRD TLI Dose Dose observationof donor type post-transplant Gender Cause (cGy) (×10⁶/kg) (×10⁶/kg)(mg/dL) Chimerism cells) 1 (37 mo) 47/M IgA 1200 11.8 3 1.7 1 mo − 2 (19mo) 24/F SLE 1200 14.5 10 1.1 12 mo + 3 (16 mo) 35/F unknown 1200 21.910 1.1 12 mo + 4 (16 mo) 33/M Unknown 1200 9 10 1.4 <1 mo − 5 (7 mo)26/F SLE 1200 7.5 10 1.1 <1 mo − 6 (4 mo) 21/M FSG 1200 7.5 20 1.3 <1 mo− 7 (4 mo) 47/F GN 1200 11.0 20 1.3 1 mo − 8 (2 mo) 26/F MGN 1200 8.8 501.3 >1 mo + 9 (2 mo) 55/M FGN 1200 11.0 50 1.5 >1 mo +

As shown by these data and in FIG. 4, in individuals receiving an HLAmismatched hematopoietic cell transplantation, stable mixed chimerismcan be obtained with a high dose of CD3+ cells, up to 50×10⁷/kg, andthis dose of T cells can overcome low numbers of CD34+ cells. Forexample, patient 7 and 9 received the same dose of CD34+ cells, but onlypatient 9, who received a high T cell dose, achieved mixed chimerism.

Example 3—Collection of Peripheral Blood Progenitor Cells for Use in aHematopoietic Cell Transplantation

To collect peripheral blood progenitor cells that are adequate inquality and quantity, which will result in acceptable progenitor cellviability and recovery for a hematopoietic transplant. This type ofprocedure may be performed on potential BMT transplant patients ordonors as well as for storage purposes.

-   -   To collect lymphocytes from a donor where the goal is to create        a graft versus leukemia effect.

To collect dendritic cells or another cell line for a research study.

-   -   For patients weighing less than 40 Kg see the Pediatric COBE®        Spectra™ Procedure for procedure location, blood or albumin        prime criteria and other age specific issues.

TABLE 2 White Blood A functionally closed COBE Spectra disposable tubingCell Set- set that allows for the addition of anticoagulant to theFunctionally product bag and in-run or post-procedure sampling. ClosedSample Bulb The sample bulb assembly consists of 2 sample bulbs Assemblywith sampling ports, a Y connector, 3 slide clamps and tubing leading tothe product bag. Sample Bulb A 4 ml plastic bulb for obtaining andremoving a product sample. Sampling Port A port connected to each samplebulb that allows removal of product sample with a blunt needle andsyringe. Accessory Line The accessory line is connected to the collectline and includes a sterile barrier filter with leer connection, afrangible, and a slide clamp. Frangible A breakable barrier thatprevents product from draining into the sterile barrier filter until thenurse is ready to add anticoagulant. Sterile Barrier A 0.2 micron filterthat allows the addition of anti- Filter coagulant to the product bagwhile maintaining a functionally closed tubing set.

Tubing Installation

Powered on the apheresis machine (e.g., Spectra) to perform a shortself-check to ensure the power supplies are operating at the correctvoltages. Verified that the “cobe spectra (version_software)” isdisplayed, the yellow warning led illuminates, the pause led flashes,the cartridge clamps are in unload position, the single stage filler isin place. The disposable set package was placed on the centrifuge coverand paper tapes removed. The package was held securely by placingunderneath the hook on front panel.

The inlet coil was removed and hung to ensure access connection on hookon left side of iv pole. The access saline (green) line was placed overthe top of the machine. The return line coil was removed and returnconnection hung on the hook on left side of iv pole. The return salineline was placed over the top of the machine. The bags were hung on ivpole from left to right: ac far left; saline; waste; plasma; product bagon right. The slide clamp was closed between the product bag and the yconnector on the sample bulb assembly to prevent product from enteringit prematurely. The return pump cartridge was removed from right side ofpackage and snapped into cartridge clamp between plasma andcollect/replace pumps.

The access pump cartridge was removed from left side of package andsnapped into cartridge clamp between ac and inlet pumps. (cobe labels oncartridges should be upright and facing out. The ac line in ac detectorwas placed and program continued to load the tubing into pumps. Thelines were placed in collect/replace and plasma valves.

The return pressure sensor was placed in its housing and pushed downwardand turned clockwise to lock in place. The rbc line was placed in therbc valve, completely inserted in rbc detector. The return and inlet airchambers were positioned in air detectors with the waste divert linesforward. Air chamber filters were positioned so the top of the chamberswere aligned with the air detector housings. The protective caps wereremoved from the male/female leer connectors above the return airchamber and the waste divert lines placed in waste divert valveassembly. The “y” was pushed in first, then “flossed” the tubing backinto place.

The line was placed in the centrifuge pressure sensor housing and theaccess pressure sensor was placed in its housing, pushed downward andturned clockwise to lock it in place. The return (blue) line was placedin return valve so line sat horizontally through center of valve. Thechannel was removed from package.

The device was unlocked, and placed in the centrifuge. The centrifugewas rotated such that the loading port was open to the front and thecentrifuge collar holder rested on outer rim of the filler. The tubingthat was connected to the channel was extended and the centrifugerotated clockwise several times to ensure tubing did not twist and upperbearing remained in place. The centrifuge door was closed and coveredprior to running a standard priming procedure for the disposable tubingset.

Connect Patient and Perform Apheresis

Using catheter for access: the catheter was clamped and the hubconnection prepped with an alcohol wipe. The needleless connector wasremoved and hub and threads cleaned vigorously with a new alcohol wipe.The vacutainer holder was attached and pinch clamp on catheter opened.The first 5 ml drawn from catheter was placed in the waste draw and thelab specimens were drawn. The pinch clamp on the catheter was closed,the attached needle removed from the spectra access line the access lineto catheter arterial line attached. Samples were evaluated forhemodilution. Access to the saline roller clamp was closed while theaccess to the line pinch clamp was opened. The program on the apheresismachine was initiated. The return line was connected to the cathetervenous line while the pinch clamps were opened and the roller clamp wasclosed on return saline line.

Using venipuncture for access: the venipuncture sites were cleaned withchlorhexidine gluconate 2% with alcohol skin prep for about 30 secondsand air dried for approximately 30 seconds. The access venipuncture wasperformed using a 17 gauge needle attached to spectra access line or 18gauge needle from stock supply. Lab specimens were drawn for possibilityof hemodilution. The return venipuncture was performed with a 20-17 giv/needle. The spectra access and return lines were connected to accessand return needles. The white pinch clamps were opened on access andreturn lines. The roller clamp was adjusted on the return saline line tokeep vein open, to prevent return needle from clotting if there would bea delay in returning through needle. Acces to the access saline rollerclamp was closed and pressure in the cuff inflated to 40 mm hg prior tocontinuing to the start run mode.

Specifications: mononuclear cells were collected using procedures at anhct of 2-4% on the colorgram (very light, 0.5-1.5% for products whichwill undergo secondary processing e.g. Isolex or clinimacs). Thecolorgram was inserted under the smallest clear collect line where itexits the centrifuge chamber, just below the multi-lumen connector. Thecolored rectangles on the colorgram to the color of the collect linewere compared. Mononuclear cell collections should have a minimum numberof red cells. Typically, there was a significant number of white bloodcells in the top layer of the red blood cells. It was necessary tocollect some red blood cells for a maximum mononuclear cell yield.However, if the product is to be further processed for cell selection,granulocytes will compete with the CD34+ cells in the CD34+ selectioninstruments. The lighter the collection, fewer granulocytes but moreplatelets were collected. If the color of the collect line is too dark,the plasma pump flow rate was decreased by 0.3 to 1 ml/minute every 3-5minutes. If the color of the collect line is too light, the plasma pumpflow rate was increased by the same adjustment.

Plasma was collected after quick start completed until the end of thetarget run time. Collecting plasma may cause temporary interfaceinstability and high return pressure alarms. In order to avid this,plasma was collected by opening the slide clamp on the plasma line,pressing the target values, pressing plasma, entering the volume ofplasma to collect and pressing enter. Near the end of plasma collection,the rate was slowed to 30-40 ml/min, as the return pressure limitallows, and then gradually increased to the previous rate.

For cryopreserved products: if the white blood cell count is greater orequal to 30 k/ul and platelet count is greater or equal to 30 k/ul onpatients, or 200 k/ul on donors, the collect rate is increased to makeproduct bag contain a minimum of 300 ml.

The slide clamp was closed above the frangible on the accessory line andthe frangible completely broken by bending the tubing containing thefrangible back and forth. Aseptic technique was used to remove the capfrom the leer connection below the sterile barrier filter and a syringecontaining the desired amount of anticoagulant was attached. The slideclamp above the frangible was opened and the anticoagulant slowelyinjected through the sterile barrier filter into the product bag. Theslide clamp on the tubing just above the frangible was closed beforeremoving the syringe to prevent back flow of fluid. A syringe containingapproximately 3 ml of preservative free saline was attached to the luerconnection below the sterile barrier filter. The slide clamp was openedabove the frangible and the saline injected through the sterile barrierfilter to flush the anticoagulant into the product bag. The slide clampon the tubing was opened just above the frangible and cap replaced.

A midbag sample was obtained by closing the slide clamp between theproduct bag and the y connector on the bulb assembly and the slide clampon the tubing between one of the sample bulbs and the y connector. Theproduct bag was mixed well to ensure a representative sample and theslide clamp opened between the product bag and the y connector on thesample bulb assembly. The sample bulb attached to the tubing wassqueezed with the open slide clamp to withdraw only the amount ofproduct sample needed. If too much product sample was withdrawn, theexcess product sample was expressed back into the product bag byinverting the sample bulb above the fluid level of the product bag,squeezing the sample bulb to express excess product sample back into theproduct bag and clearing the tubing of product sample using residualair, by holding the sample bulb upright and below the product bag,squeezing the sample bulb using residual air in the sample bulb to pushproduct sample from the tubing into the product bag and whilemaintaining pressure on the sample bulb, closing the slide clamp justbelow the y connector.

The product sample was aspirated from the sampling port with a blunt 18gauge 1 inch needle and a syringe by inserting a blunt needle withattached syringe into the sampling port, inverting the sample bulb,slowly aspirate the product sample into the syringe, removing the needlewith attached syringe from the sampling port and transferring theproduct sample to test tube.

Rinseback

Start rinseback using catheter for access: the roller clamp on greensaline line was opened and the access catheter flushed with 10 ml ofsaline, then the white pinch clamp on access line was closed. The returnline was flushed with 10 ml of saline and the white pinch clamp onreturn line was closed. The roller clamps on saline lines were closedand the connection sites cleaned with alcohol. The cathether was clampedand the access line closed before attaching a new needleless connectorpre-filled with heparin. The lumen was flushd with 3 ml, 100 units/mlheparin and the syringe and reclamp catheter were removed. The sameprocedure was repeated for the return line.

Start rinseback using venipunctures for access: the pressure on cuff wasreleased and the roller clamp on green saline line opened tto flushneedle, then the white pinch clamp on access line closed. The draw lineneedle was removed. The white pinch clamp on return line was closed andthe return needle removed. Pressure was applied to the venipuncturesite, and the roller clamps on saline lines closed.

Example 4—Isolation and Purification of CD34+ Cells from ApheresisProducts

The CliniMACS system was used for the selection and enrichment of CD34+hematopoietic progenitor cells (HPC). The CliniMACS system employs CD34antibodies conjugated to magnetic particles as a means of labeling thetarget progenitor cells. Once labeled with these magnetic particles, thecell suspension was passed through strong magnetic gradients within theselection column on the CliniMACS device. The magnetically-labeled CD34+cells were retained within the column while unlabeled cells flowedthrough the column and were collected in the Reduced Fraction bag. Whenthe column was removed from the magnetic field, the CD34 positive cellsare released for collection in the Enriched Fraction bag.

Reagent Preparation

TABLE 3 Final Concen- Solution Reagents Volume tration Storage CliniMACSPBS Buffer 1 L 0.5% HSA in 1 bag COBE Buffer Containing 1 CliniMACS 1bag mM EDTA Buffer CliniMACS pH 7.2 25% HSA 20 ml 1 bag BSC/ COBE HSA/Normosol 46 ml 2% HSA/ Normosol Normosol Formulation 25% HSA 4 ml MediaCryo- Hetastarch 12.2 ml 1.8% HES 4 C. Fridge protectant 25% HSA 4.8 ml2.5% HSA Media DMSO 3 ml 7.5% DMSO 4 C. Fridge

Product Preparation

A settle plate was placed on each side of the biosafety cabinet andproduct brought into the cabinet. For HPC-Apheresis products test wasperformed on each product prior to pooling. A 3-way sampling transferset was inserted into the bag. For products that will be stored in therefrigerator overnight, autologous donor plasma was added to yield acell concentration of <200×10E6/ml. On the following morning, theproduct equilibrated at room temperature before processing to preventclumping.

The cells were transferred from the apheresis collection bag to the 600ml transfer pack labeled as “cell preparation” (heat seal leaving 6-8inches of transfer pack tubing for sterile docking later). The productwas weighted and the product volume calculated.

Removal of excess plasma when product is >200 ml: the product wasbalanced and centrifuged at 250 g for 15 minutes with no brake at roomtemperature. A sterile product bag was docked to the 600 ml transferpack labeled “waste plasma” and tubing clamped with a hemostat. Theproduct bag was removed from the centrifuge bucket to avoid disturbingthe cell sediment and hung on the plasma extractor. The plasma extractorhandle was used, the hemostat opened and plasma expressed into the wastebag. The tubing was clamped with the hemostat to stop the flow. The cellbag was removed from the plasma extractor and the cell pelletresuspended. The cells were placed on the tared scale and both air andplasma expresesd back into the cell bag until bag volume was <200 ml.The hemostat was closed and cell pellet resuspended in the cellpreparation bag by gently swirling the bag. Proceed to Platelet Wash.

Platelet wash when product is <200 ml: a plasma transfer set wasattached to one of the prepared buffer bags and the tubing of thetransfer set was sterily docked to the cell bag. The roller clamp wasopened and the Cell Preparation bag filled with buffer. The tubingbetween the Cell Preparation bag and the buffer bag was heat sealed with6-8 inches of room. The remainder of the buffer bag was retained forRemoval of Excess Reagent. The centrifuge was balanced and bagscentrifuged at 250 g for 15 minutes at room temperature, no brake. Theproduct bag was sterily docked to the 600 ml transfer pack labeled“Platelet Waste” and tubing clamped with a hemostat. The product bag washung on plasma extractor pins and plasma released from the plasmaextractor handle, the hemostat opened and plasma expressed into thewaste bag. The tubing was clamped with a hemostat to stop the flow andthe cell bag removed from plasma extractor and cells resuspended. Theproduct bag was placed on a tared scale and adjusted to the appropriateantibody incubation weight (see CD34 reagent weight parameter tablebelow). The total nucleated cell and/or CD34+ cell content and productand reagent volumes were within the specified ranges for optimallabeling of the cells.

TABLE 4 TNC and CD34 <60 × 10⁹ TNC or >60 × 10⁹ TNC or limitations <600× 10⁶ CD34 >600 × 10⁶ CD34 Incubation Weight 95 g (90-100 g) 190 g(180-200 g) CliniMACS CD34 1 vial (7.5 ml) 2 vials (15.0 ml) Reagent

CliniMACS Set-up

The tubing set was prepared inside the biological safety cabinet and the“CD34 Enriched” collection bag: heat sealed. 150 ml was removed from atransfer bag and a recipient bag attached, the blue pin inserted andconnected to the CliniMACS luer connector. A Pall filter was insertedvia the blunt end to the CliniMACS bubble trap spike. The CliniMACSdevice was powered on and self-diagnostic procedures run.

Priming

The CliniMACS system automatically primed once the program wasinitiated. Buffer circulated through the tubing set and collected inboth the priming waste bag and buffer waste bags.

CD34 Reagent Labeling

The total cell number was counted and required number of vial(s) of CD34Reagent added to the product bag based on the total number of cellscollected. The contents of the cell preparation bag were mixedthoroughly using a gentle end-over-end motion at 5-10 minute intervalsor the bag was placed on a rocker at 25 rpm and angle 10% for 30minutes.

Excess reagent was removed by repeating the steps in the “platelet washsection” except that two transfer bags were labeled, one ‘Reagent Wash1’ and one ‘Reagent Wash 2’, to substitute Platelet Waste bags.CliniMACS Buffer was added and the wash performed. The product wasweighted on a tared scale and product weight adjusted to the The StartProduct weight range of between 80 g and 318 g.

CD34 Selection

A Start Product Bag was connected to the CliniMACS and the cap from thetop of the Pall filter was removed to spike the Start Product bag. TheStart Product bag was placed on the center hanger bar and the CD34+selection procedure initiated by pressing RUN on the CliniMACS. Theselection process is automated and lasted about 40 minutes.

Cell Formulation

A sample from the Reduced Fraction Bag was taken to determine cellcount, viability, sterility, and perform FACS assays for CD3+ cellcounts.

Cryopreservation

The centrifuge was pre-cooled to 4° C. and a sample from the CD34Enriched Fraction Bag taken to determine cell counts, viability, anycolony forming units, and flow cytometry assays. The contents of theCD34 Enriched Fraction bag were transferred equally between two labeled50 ml conical tubes. The CD34 Enriched Fraction bag was rinsed withNormosol/2% wash mixture and divided between the two tubes. Centrifugetubes at 1700 rpm for 8 minutes, 4° C. on low brake. The supernatant wasremoved and transferred to a sterile waste tube(s), the pellets wereresuspended by gentle tapping. The supernatant was used for a cellcount, sterility and endotoxin testing.

4 ml Normosol/2% HSA was added to one of the tubes, the contents mixedand transferred to the other tube. The first tube was rinsed with 2 mlNormosol/2% HSA and transferred into the other tube, the cell volumeadjusted to 7.8 ml and the cell suspension cooled in the refrigeratorfor 5-30 minutes.

The controlled rate freezer was prepared for a controlled rate freezeaccording to the manufacturer's protocols and further explained inExample 4 below. Once the controlled rate freezer was at temperature ofless than −6° C., 7.8 ml of cryoprotectant solution was added to thecells. 15 ml of cell/cryoprotectant was transferred to a 50 ml cryobagand heat-sealed. The sample probe was attached to the center of thecryobag with a piece of tape and the bag positioned in the press so thatthe ports protruded from the top of the press. The press was closed andthe labeled cassette placed into the controlled rate freezer ahead ofactivating the controlled rate freezer run.

Example 5—Cryopreservation of CD34+ Cells from Apheresis Products

Optimal viability of frozen therapeutic cells is achieved usingcryoprotectant and a slow rate of cooling. A freezing solution usingDMSO as a cryoprotectant is added to the cellular product to preventdamage to the cells caused by ice crystal formation. The CBS controlledrate freezing system consists of a freezing chamber, notebook PCequipped with Microsoft Windows operating system, Cryogenic FreezingSoftware, sample probe, sample racks, and LN2 transfer hose. A printerhas been added so that each product may have a copy of the freezingcurve for its respective run, to include in the processing record. TheCRF freezes at pre-defined user programmable rates. A thermocouple probeis attached to one of the cryobags or inserted into a monitor cryovial.The chamber temperature is continuously monitored. Advancement of thecryopreservation profile steps occur as a thermocouple registerspre-defined target temperatures. When the final chamber temperaturespecified by the profile is achieved, an alarm sounds and the product isremoved and placed into a permanent storage freezer. Audible and visualalarms occur if a deviation between chamber and programmed targettemperatures is detected.

Cryoprotectant Preparation

The appropriate cryoprotectant solution was prepared as described in thetable below. Cryoprotectant solution was chilled for at least 30 minutesat 4-6° C. before use.

TABLE 5 Washed Products (enriched or depleted Unmanipulated products -not containing HPC/TC (50 or plasma) (1 ml vial or 100 ml total volume)15 ml bag total volume) Cell suspension:cryoprotectant ratio 70:30 50:50Final conc. Final Conc. Components Volume In product Volume In ProductDMSO   10 ml 10%   3 ml 7.5%  Normosol 20.4 ml 20% HSA (25%)  4.8 ml 4%Hetastarch 12.2 ml 3% Total 30.4 ml 20.0 ml

Cryopreservation Container Selection

The appropriate cryopreservation container to use is based on celldensity, product type, volume and individual protocol requirements. Perindividual processing procedures, the appropriate freezing container andpreparation of cell/cryoprotectant volumes was performed per the below.

TABLE 6 Freeze Mix # Freezing Initial Product TNC Prefreeze VolumeVolume Bags/vials TNC ≤ 3 × 10⁹ 35 ml 15 ml 1 (250 ml) TNC > 3-≤25 × 10⁹35 ml 15 ml 1 (250 ml) (pediatric patients < 50 kg) TNC > 3-50 × 10⁹ 70ml 30 ml 1 (500 ml) TNC > 50-100 × 10⁹ 140 ml 60 ml 2 (500 ml) TNC >100-150 × 10⁹ 210 ml 90 ml 3 (500 ml) TNC > 150-200 × 10⁹ 280 ml 120 ml4 (500 ml) TNC > 200-250 × 10⁹ 350 ml 150 ml 5 (500 ml) TNC > 1-100 ×10⁶ 0.5 ml 0.5 1 ml per 1.8 ml vial

Addition of Cryoprotectant to Product

Once the controlled rate freezer reached temperature to receiveproducts, the freezing media was added to the product(s). An appropriatevolume of cryoprotectant was added to each vial or bag according toindividual processing procedures. Optimal viability was achieved whentime between the addition of cryoprotectant and placement of the productinto the CRF was minimized. Cryobags were heat sealed and excess tubingremoved.

The bag(s) were placed in an appropriate storage cassette and eachcassette placed into the controlled rate freezer. For 250-0500 mlcryobags, one of the products frozen was placed in a cassette containingthe monitoring probe. The cassette prepared for the long term storage ofthis product was placed in the controlled rate freezer by the monitorcassette, so that the frozen product was transferred into the coldcassette at the end of the run.

Chamber Preparation

The appropriate rack was placed into the chamber, large cassette rackfor products in 250-500 ml cryobags, abag press for 50 ml cryo bags or acryovial rack for products in cryovials. For 250-500 ml cryobags asample probe was attached to a cassette with tape so that the protrudingside of the probe end was facing up and positioned such that it came incontact with the center point (fullest part) of the cryobag. Forcryovials, the needle thermocouple probe was inserted through a rubberstopper to seal the monitor cryovial. For small 50 ml cryobags, a sampleprobe was taped directly to the center of the product before it wasplaced into the bad press. The sample probe connection is located on theinside of the chamber in the upper left hand corner of the fan guard.The probe was plugged into the jack and the chamber door secured.

Cryopreservation

Once the chamber reached ≤6.5° C., the product(s) prepared forcryopreservation were added to the chamber. For 250 and 500 ml bags, oneof the products was placed into the cassette with the attached probe sothe probe pressed against the center (fullest part) of the back of thecryobag, and closed it into the cassette. For 50 ml cryobags, the probewas attached to the center back of the bag with a piece of tape, andpositioned the product in the bag press so that the ports protrude fromthe top of the press. The press was closed and a second 50 ml bag wasplaced in a second press and stacked on top if necessary. The freezingprogram was initiated per standard procedures.

Example 6—Thawing and Washing Cell Products

This example applies to HPC/TC products that have been collected andprocessed in-house or collected, processed and shipped by an outsidefacility for a recipient at SHC or LPCH. Upon the request of the medicaland/or lab director and the attending physician, the lab will thaw andwash the cryopreserved product in the lab prior to product infusion. Thethawed product is washed with Low Molecular Weight Dextrose and Albumin.Washing the product is done to remove excess hemoglobin for HPC/TCproducts that exceed red cell volume limitations, to reduce DMSO volumeespecially for small recipients (<50 kg), to remove excess plasmaproteins in the allogeneic setting, and to improve the recovery ofviable cells.

Reagent and Equipment Preparation—the Day Before the Infusion

The 10% LMD, ACD-A, and 5% HSA were placed in a monitored refrigeratorand sufficient cold gel mats were placed in the refrigerator. Thewaterbath was prepared.

Reagent and Equipment Preparation—the Day of the Infusion

The water bath, BSC and processing suite were prepared and thecentrifuge cooled down to 6° C. for 30 minutes. Two cold gel mats werecleaned with IPA wipes and each placed inside a sterile plastic bag. Thebags were sealed and returned to the monitored refrigerator. Thetransport cooler and a medium sized frozen gel pack were cleaned withIPA wipes

Media Preparation

Thawing Media:

The tubing on the 150 ml transfer bag labeled “Thawing Media” was heatsealed and a blue dispensing pin attached to one of the ports. A bluedispensing pin was inserted into the appropriate port of the 10% LMDbag. A 60 ml syringe was used to withdraw 60 ml of 10% LMD andtransferred to the Thawing Media bag. A mini-spike was inserted into theport of the 5% HSA bottle. A 60 ml syringe was used to withdraw 60 ml of5% HSA bottle and transferred to the Thawing Media bag. A bluedispensing pin was inserted into the appropriate port of the ACD-A bag.A 12 ml syringe was used to withdraw 12 ml of ACD-A and transferred tothe Thawing Media bag. The Thawing Media was mixed thoroughly.

Reconstitution Media:

The tubing on the 150 ml transfer bag labeled “Reconstitution Media” washeat sealed and a blue dispensing pin attached to one of the ports. A 60ml syringe was used to withdraw 60 ml of 10% LMD and transferred to theReconstitution Media bag. A 60 ml syringe was used to withdraw 60 ml of5% HSA and transferred to the Reconstitution Media bag. Mix theReconstitution Media thoroughly.

Product Thaw

The product was thawed only to the point that it was still “slushy”;some presence of ice is acceptable. The product bag was wiped with IPAwipes and placed inside the BSC. The product was wrapped in the cold gelmat in the BSC, the product kept chilled during the remaining processingsteps

Products equal to or less than 30 mls: a Cell Wash/Infusion bag set oftwo bags and three tubing sections was used for these volumes. Thetubing with the 2 spikes connected to the cryobag, the tubing with theblue luer adapter connected to the syringe containing the thawing media.The washed product was collected and centrifuged in the bag labeled‘Cell Wash/Infusion bag’ and the supernatant was be transferred to theremaining bag. The wash/infusion set clamps were closed and the outsideport covers of the cryobag with cleaned with sterile alcohol pads.

The bag was held upright and for each port, the hard part of the portwas held with the thumb and forefinger of one hand. With the other hand,an infusion set spike was inserted into the disinfected port andsecured. Thawing Media was sterily added to the bag by releasing theclamps on the tubing between the Thawing Media syringe and the cryobag.Half of the Thawing Media was transferred into both cryobag compartmentswith gentle mixing.

The clamp to the Cell Wash/Infusion bag was opened and the diluted cellsuspension transferred from the cryobag into the Cell Wash/Infusion bag.The clamp on the Cell Wash/Infusion bag was closed for rinsing process.Small amounts of the remaining Thawing Media were added to rinse bothcompartments of the cryobag and transferred the rinse to the CellWash/Infusion bag. The mixture equilibrated for 5 minutes.

An aliquot of 10% LMD was added to the cryobag, the cryobag rinsed andthe rinse transferred to the Cell Wash/Infusion bag. The above step wasrepeated until an appropriate amount of 10% LMD was dispensed into thecryobag.

The Cell Wash/Infusion bag was placed in a plastic centrifuge bag, thecentrifuge balanced and the product centrifuged at 400×g for 20 minutesat 6° C. with low brake. The bag of diluted product was placed in theplasma expressor and the supernatant slowly expressed into the attachedsupernatant bag, the flow rate controlled with a hemostat. A dispensingpin was inserted into one of the ports of the Cell Wash/Infusion bag andthe cell pellet massaged into resuspension. Reconsitution media wasadded to the Cell Wash/Infusion bag and the contents thoroughly mixed.

Products Greater than 30 mls:

A dispensing pin was inserted into one of the ports of the cryobag and asyringe containing the Thawing Media was attached to the dispensing pin.Half of the Thawing Media was transferred into the cryobag, mixed, andthe cryobag spiked with a 300 or 600 ml transfer pack, the contents ofthe cryobag transferred to the transfer pack. The cryobag was rinsedwith the remaining Thawing Media and added to the transfer packcontaining the cells, the mixture equilibrated for 5 minutes.

An aliquot of 10% LMD was added to the cryobag, the cryobag rinsed andthe rinse transferred to the Cell Wash/Infusion bag. The above step wasrepeated until an appropriate amount of 10% LMD was dispensed into thecryobag.

The Cell Wash/Infusion bag was placed in a plastic centrifuge bag, thecentrifuge balanced and the product centrifuged at 400×g for 20 minutesat 6° C. with low brake. The bag of diluted product was placed in theplasma expressor and the supernatant slowly expressed into the attachedsupernatant bag, the flow rate controlled with a hemostat. A dispensingpin was inserted into one of the ports of the Cell Wash/Infusion bag andthe cell pellet massaged into resuspension. Reconsitution media wasadded to the Cell Wash/Infusion bag and the contents thoroughly mixed.

Post Thaw Counts

The supernatant was mixed thoroughly and a sample used for a cell count.The supernatant bag was weighted and the supernatant TNC calculated.

Cells for Infusion

Two samples were removed for analysis, 1 mL for Flow analyses and a 0.2ml for a nucleated cell count, CFU and viability. The infusion bag wasweighed and placed in a sterile plastic bag and stored in a transportcooler with frozen gel pack until infusion. Post thaw TNC and % TNCrecovery were calculated and cells re-spun per table 6 below.

TABLE 7 Supernatant Infusion Cells % NC × 10E06/ml TNC Recovery Re-spinSupernatant ≤2 ≥80% no ≤2  <80% Ask supervisor/lab director >2  <80%yes >2 ≥80% no

Re-Spin the Supernate

The supernate was transferred to another transfer bag and centrifuged at400×g for 20 min at 6° C. with low brake. The bag was then placed in theplasma expressor and a blue dispensing pin inserted into one of theports of the bag. A luer connector was attached to the pin of thetransfer bag to the dispensing pin and the expressor handle released toexpress off the supernatant, the flow rate controlled with the hemostat.5 ml of each 10% LMD and 5% HSA were added to this second cell bag, thecells resuspended and the recovered cells added to the initial cellinfusion bag.

A sample was removed for a nucleated cell count and viability, Flowanalyses, and CFU assay from the cell infusion bag. The infusion bag wasweighed, the volume calculated and recorded.

Calculations

%  TNC  Recovery = post  thaw  TNC  (cells) ÷ [Post  thaw  TNC  (cells) + supernatant  TNC] × 100${{Total}\mspace{14mu} {CFU}\mspace{14mu} {Count}\mspace{14mu} X} = \frac{{TNC} \times \left( {{colonies}\mspace{14mu} {counted}\mspace{14mu} {per}\mspace{14mu} 10E\; 05\mspace{14mu} {cells}\mspace{14mu} {plated}} \right)}{10E\; 05}$${{CD}\; 34\mspace{14mu} {cell}\mspace{14mu} {count}\text{/}\mu \; l} = \frac{\left( {{NC} \times 10E\; 06\text{/}{ml}} \right) \times \% \mspace{14mu} {CD}\; 34}{1000}$

Infusion

The infusion cell bag was removed from the prepared transport coolerwith frozen gel pack and the thawed product was taken to the unit.

Example 7—Hematopoietic Progenitor Cell and Therapeutic Cell Infusionsfrom Fresh or Cryopreserved Allografts or Autografts

Allograft (allogenic) transplant involves infusing donor bone marrow,hematopoietic progenitor cells (HPC) or therapeutic cells (TC) into arecipient who is genetically different. Matched or partially matchedfamily members or unrelated donors can be used as donors. Thehematopoietic cells are collected and then infused into the recipientwithin 24 hours. Therapeutic cells are collected and infused into theusually within 24 hours. Alternatively, hematopoietic cells maybecollected and cyropreserved.

Unrelated umbilical cord blood (UCB) is a valuable sources of donorcells for HPC transplantation, thus extending this treatment modality topatients who lack other donors.

Autograft (Autologous) transplant involves the recipient receiving hisor her own bone marrow or hematopoietic progenitor cells (hpcs).

If a large volume of hpcs (more than 4 bags) are to be infused,physician may stop after half of the bags and resume transplant 1 to 2hours later. This is to prevent circulatory overload due to the largevolume of fluid infused in a short period of time. Check with physicianto determine if patient should be remedicated prior to resumingtransplant.

TABLE 8.1 NURSING ACTIONS KEY POINTS Pre-administration: A. The sourceof the graft was determined and Urine may be pink in color for 24 hoursdue to the method of infusion explained to the patient. The culturemedia that was added to hpcs and/or possible side effects were describedand patient hemolysis of red cells. instructed to notify RN if anyoccur. Pre-medication was required. If ABO 1. Administration checks aresimilar to other incompatible, red cells must be removed from bloodproducts. the graft. 2. Check ABO compatibility for allogeneic patients.B. The approximate time of the planned infusion Premedications mayconsist of diphenhydramine was determined. The patient was premedicatedas (Benadryl) and hydrocortisone to minimize ordered prior to thetransplant. possible reactions. C. O₂ with nasal cannula and suctionwere setup In the event of an allergic reaction or fluid in room.diphenhydramine (Benadryl) and overload. furosemide (Lasix) were readilyavailable (in the Pyxis Station). Baseline vital signs were establishedwithin one hour of infusion. D. Using a primary and secondary IV tubing(no filter), the NS was connected to prime the primary tubing as thebackup. Back flush to prime the secondary tubing for which the cellswill infuse. A new secondary tubing for each bag of cells to be infusedwas obtained. E. The large lumen of central venous catheter Patency ofline assessed before infusion of cells. (CVC) was used to removeneedleless connector and Line must run wide open by gravity. If neededthe IV tubing attached directly to the hub of the large be prepared tostart a peripheral IV. lumen. F. MD injected anticoagulant intocryopreserved product to prevent cells from clotting. G. The product wasbrought to the bedside by Cryopreserved hpcs were infused within 10-15the MD. (This product is NOT to be irradiated or minutes of thawing topreserve cell viability. filtered.) The cells were completely infusedwithin 30 minutes of thawing.

TABLE 8.2 Administration: A. Fresh (not cryopreserved) marrow, HPC orTC: 1. A spike of secondary tubing was inserted into the bag of cells.The IV pump was programmed for the appropriate time and volume. 2. Cellswere infused at 250 ml/hr. 3. There may be 1 or 2 bags of cells. Thesecond bag of cells are not released until infusion of the first bag iscomplete. Cell infusion length may vary due to total volume of cells 4.Vital signs were documented15 minutes after Monitored for signs andsymptoms of an allergic start of infusion. reaction. Cryopreserved HPCor TC: 1. spike of secondary tubing was inserted into the bag of cells.The IV pump was programmed for the appropriate time and volume 2.Cryopreserved cells were infused over 10 To maintain cell viability, thecells were minutes. completely infused by 30 minutes of thawing. 3.After infusing cells, the infusion tubing was CD 34 selected cells wereflushed with a syringe rinsed and the bag emptied with a backflush ofNS. of 2% human serum albumin and normalsol. The remaining cells wereinfused using this back flush method. 4. If a large volume of hpcs (morethan 4 bags) Large volume of fluids infused over a short are to beinfused, repeat vital signs and lung period of time may cause fluidoverload. assessment half, way through infusion and as Attendingphyscian must be in direct supervision clinically needed. of infusion ofTC. C. Cryopreserved/frozen Therapeutic cells: 1. If cells are in asmall volume (e.g. Tcs), cells were be administered using a syringe bydirect IV push method at catheter hub. Flush catheter before and aftercell infusion with saline.

TABLE 8.3 Post-administration: A. The patient's, respiratory status andskin were Signs and symptoms of reaction may include assessed forpossible reactions at the end of the nausea, rigors, pulmonary emboli,shortness of infusion. The vital signs were assessed again breath,tachycardia, bradicardia (with frozen within 1 hour post infusion.cells) and cardiac overload. C. At the end of the infusion, a newneedless connector was set and then heparin-lock the central catheter.The previous solution infusion may also be resumed.

What is claimed is:
 1. A cellular product for establishing mixedchimerism in a solid organ transplant recipient, the product comprising:at least 5×10⁶ CD34⁺ cells/kg recipient weight; and a defined amount ofabout 5×10⁶ CD3⁺ cells/kg recipient weight, wherein the cellular productis substantially devoid of platelets and red blood cells.
 2. Thecellular product of claim 1, comprising 5×10⁶ CD34⁺ cells/kg recipientweight.
 3. The cellular product of claim 1, comprising greater than5×10⁶ CD34⁺ cells/kg recipient weight.
 4. The cellular product of claim1, wherein the CD34⁺ cells have been enriched.
 5. The cellular productof claim 4, wherein the CD34⁺ cells have been column enriched.
 6. Thecellular product of claim 1, wherein the CD34⁺ cells and the CD3⁺ cellsare provided in separate containers.
 7. The cellular product of claim 1,wherein the CD34⁺ cells and the CD3⁺ cells are provided as a mixture ina common container.
 8. The cellular product of claim 1, wherein theCD34⁺ cells and the CD3⁺ cells are HLA matched to an HLA type of thesolid organ transplant recipient.
 9. The cellular product of claim 1,wherein CD34⁺ cells and the CD3⁺ cells are from a single apheresisproduct.
 10. The cellular product of claim 1, further comprising acryopreservation solution.
 11. A cellular product for establishing mixedchimerism in a solid organ transplant recipient that is HLA matched tothe solid organ transplant donor, the product comprising: at least 5×10⁶CD34⁺ solid organ transplant donor derived cells/kg recipient weight,wherein the solid organ transplant donor's CD34⁺ cells are of an HLAtype that is matched to the solid organ transplant recipient's HLA type;and a defined amount of about 5×10⁶ CD3⁺ cells/kg recipient weight,wherein the solid organ transplant donor's CD3⁺ cells are of an HLA typethat is matched to the solid organ transplant recipient's HLA type,wherein the cellular product is substantially devoid of platelets andred blood cells.
 12. The cellular product of claim 11, comprising 5×10⁶CD34⁺ cells/kg recipient weight.
 13. The cellular product of claim 11,comprising greater than 5×10⁶ CD34⁺ cells/kg recipient weight.
 14. Thecellular product of claim 11, wherein the CD34⁺ cells have beenenriched.
 15. The cellular product of claim 14, wherein the CD34⁺ cellshave been column enriched.
 16. The cellular product of claim 11, whereinthe CD34⁺ cells and the CD3⁺ cells are provided in separate containers.17. The cellular product of claim 11, wherein the CD34⁺ cells and theCD3⁺ cells are provided as a mixture in a common container.
 18. Thecellular product of claim 11, wherein the CD34⁺ cells and the CD3⁺ cellsare HLA matched to an HLA type of the solid organ transplant recipient.19. The cellular product of claim 11, wherein CD34⁺ cells and the CD3⁺cells are from a single apheresis product.
 20. The cellular product ofclaim 11, further comprising a cryopreservation solution.